CN104919055A - Methods and compositions for detecting multiple analytes with a single signal - Google Patents

Abstract

Compositions, methods, and devices for the detection of multiple analytes with a single signal are provided.

Description

The method and composition of multiple analytes is detected with single signal

Technical field

Embodiment partly detects multiple analytes for single signal.

Embodiment

Before description composition provided herein and method, should be appreciated that embodiment is not limited to described ad hoc approach, composition or method, because it can change.Should also be clear that term used in description is the object for describing some embodiments, and be not intended to the category limiting embodiment.

Various method and embodiment are described herein.Method and embodiment can combinations with one another.Definition described herein and embodiment are not limited to ad hoc approach or example, and it should be so limited unless the context clearly dictates.

As used herein, phrase " detection of analyte ", " detect analytes " refer to the detection carrying out multiple analytes with single signal.The detection of multiple analytes can be to detect at least with single signal as described herein or just in time 2,3,4 or 5 kind of analyte.

Must be noted that, as herein and in following claims use, unless the context clearly dictates otherwise, otherwise singulative " (a) ", " a kind of (an) " and " this (the) " comprise and multiplely mention thing.Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with those skilled in the art usually understand identical implication.Although any method being similar to or being equivalent to method described herein may be used to practice or test embodiments of the present invention, now preferred method is described.The mode that all publication are herein quoted all is in full incorporated herein, the theme that its incorporated extent support is described at present.Should not be construed as absolutely herein and admit that theme is had no right prior to disclosure due to prior inventions.

As used herein, term " about " mean the numerical value of the numeral therewith used ± 10%.Therefore, about 50% mean within the scope of 45%-55%.In addition, phrase " about X to Y " is identical with " about X is to about Y ", and that is term " about " is modified both " X " and " Y ".

As used herein, structure, event or situation that term " optional " or " alternatively " mean to describe subsequently can or can not occur, and describe and comprise the situation and event situation about not occurring that event occurs.

As used herein, term " sample " means to contain special article (such as analyte) or doubtful any fluid medium or the liquid containing special article.In some embodiments, the sample that is rich in dissolved solids can be used and without the need to further process, and in some embodiments, the sample containing high solid (non-dissolving) can be analyzed by using strainer or be used in conjunction with other manual step.Sample in for method described herein or device before also can be non-filtered or purifying.Sample can be the sample of liquid, suspension, extraction or dissolving, or supercutical fluid.If sample will be used for flow-through appt (vertical or horizontal (lateral)), so some flow characteristicss must be present in sample or extract to allow to flow through device described herein and system.The example of sample includes, but is not limited to blood, food swab, food extract, food suspension, food culture, bacterial cultures, viral cultures, amplified reaction, saliva, biofluid, PCR reaction etc.Sample can also obtain from another sample.For example, can to carrying out PCR reaction from the nucleic acid mixture of another sample (such as food, cell, virus, bacterium, blood etc.) extracting and developing and/or purifying.PCR reaction will be regarded as the sample obtained from another sample.

" food suspension " refers to the food eaten raw or boiled placed or be suspended in solution.Food solns can for mixing, vortex or fusion." food culture " foodstuff samples for cultivating under the condition of enriched sample.This method also can be called " enrichment ".Enrichment may be used for promoting that sample analysis is to detect the presence or absence of multiple analytes better with single signal.Sample also can be the response sample derived from different sample.The example of response sample is " enrichment ".For example, blood or foodstuff samples can treated (such as, cultivation, purifying, be separated into component etc.) and can test treated sample to detect multiple analytes.In some embodiments, in blood sample or foodstuff samples, two kinds of analytes are detected.In some embodiments, can by carrying out two kinds of specific two kinds of amplified reactions of analyte tool and two kinds of amplified productions can be detected with single signal subsequently carrying out detect analytes with the existence detecting two kinds of analytes in sample concurrently.In some embodiments, single signal is used to detect three kinds of analytes.Detection for what walk abreast, that is, only can produce signal when all analytes are all present in same sample.Parallel signal generation can be realized by producing bridge joint mixture described herein.The non-limiting embodiment of bridge joint mixture is found in Fig. 1-Fig. 3.

As used herein, term " solid support thing " means material substantially insoluble in selected system, or easily can be separated the material of (such as, by precipitation) from the system selected by it is present in wherein.The solid support thing being applicable to put into practice the inventive method can comprise activated maybe can activation and allow some compound or molecule (such as capture agent, antibody etc.) to be incorporated into the group of solid support thing.Solid support thing can be, such as, agarose (agarose), sepharose (sepharose), polyacrylamide, agarose/polyacrylamide copolymer, dextran, Mierocrystalline cellulose, polypropylene, polycarbonate, soluble cotton, glassine paper maybe can provide any other suitable substance of appropriate solid upholder.In some embodiments, solid support thing can for being applicable to the form of chromatographic particle, powder or gel.Solid support thing also can be film, such as soluble cotton, PVC etc.Also can use the film of other types, and there is no specific requirement about the type of operable film.In some embodiments, solid support thing is test membrane.The example of test membrane is described in herein.

As used herein, term " analyte " includes, but is not limited to antigen, nucleic acid molecule, amplified production (such as amplicon), peptide, sugar etc. by the microorganism encodes of cell, virus, bacterium or other types.In some embodiments, analyte is not antibody or its function fragment.Can as described herein by using method described herein and other known methods or device such as amplification method (such as PCR, RT-PCR etc.), hybridizing method, primer etc. through marking to combine to detect nucleic acid molecule.Term " target molecule " can exchange with term " analyte " and use.Amplification method may be used for the amount of the nucleic acid molecule existed in amplification sample to promote the detection of analyte.The analyte of the other types that method described herein can be used to detect includes, but is not limited to antigen, antibody, acceptor, part, inner complex, protein, enzyme, nucleic acid, DNA, RNA, Insecticides (tech) & Herbicides (tech), inorganic or organic compound, maybe can find any material of specificity combinating reagent.Analyte also can refer to the different epi-positions be present on same protein or polypeptide.Analyte also can refer to the analyte from cause of disease or nonpathogenic organism body.Analyte also can be called the analyte paid close attention in sample.That is, analyte can be called and just determines the reagent of presence or absence in sample by user.

As discussed herein, analyte can be amplified production, as the product of PCR reaction.PCR primer is the nucleotide sequence that amplification carrys out test sample.Therefore, the PCR primer detected in sample determines whether the nucleotide sequence as the basis of PCR primer is present in initial sample.For example, if those skilled in the art is just determining whether foodstuff samples is subject to e. coli contamination, so can increase (such as, by PCR) the specific nucleotide sequence of intestinal bacteria tool is detected according to method described herein subsequently.The detection instruction of amplified production (that is, amplicon), foodstuff samples contains the specific native sequence nucleic acid of intestinal bacteria tool.This example is nonrestrictive and may be used for the analyte detecting other nucleotide sequences or the other types existed in Natural Samples.Analyte can for exist in initial sample or be the analyte by such as using PCR to obtain from initial sample.When just detecting multiple analytes according to method single signal provided herein, analyte also can have allos label (tag) or interaction unit, and modified analyte is also referred to as analyte.In some embodiments, analyte will not contain allos interaction unit, as fluorescence labels, vitamin H, digoxin etc.

Analyte is different from the presence or absence reagent for detect analytes.Therefore, add in sample to determine that the reagent whether analyte exists is not paid close attention to analyte.Such as, in typical sandwich assay, the first antibody is connected to solid support thing.Be coated with the solid support thing of antibody and sample contacts to determine to be incorporated into the presence or absence of the antigen of antibody.Also add secondary antibody (secondary antibody) subsequently with detectable antigens.Usual by adding three grades of antibody (the 3rd antibody subsequently, third antibody) detect the existence of secondary antibody, three grades of antibody have the enzyme being such as incorporated into it, and it can be detected by various mode (antibody that such as HRP connects).Secondary antibody is not paid close attention to analyte, because it is the reagent for detection of primary antigen (primary antigen).Therefore, sandwich assay can not detect the existence of multiple analytes according to method single signal described herein, because secondary antibody is detectable antigens or the presence or absence reagent of analyte paid close attention to or instrument.Analyte neither be found in component on bridge joint entity or part.Such as, in U.S. Published Application US 2010/0273145, Fig. 1 and Fig. 2 display analysis thing is incorporated into bridge joint entity, and bridge joint entity is incorporated into signalling (signaling) entity subsequently with the existence of detect analytes.The analyte that bridge-jointing unit or its any part or signalling entity are not analyte or pay close attention to.These components are the reagent for detect analytes, and when U.S. Published Application US2010/0273145, this is the detection of single analyte.U.S. Published Application US2010/0273145 is incorporated to thus by reference about the explanation of its figure and its component.

In some embodiments, analyte is protein, as pathogen protein matter.Pathogen protein matter refers to the protein from pathogenic agent.The example of pathogenic agent includes, but is not limited to virus, prokaryotic organism body and such as pathogenicity bo most eukaryotes, as unicellular Pathogenic organisms and many cells parasite.Pathogenic agent also can comprise protozoal pathogens, and it comprises in life cycle, and it is the stage of intracellular pathogen.As used herein, term " intracellular pathogen " means virus or Pathogenic organisms, and being present at least partially in host cell and producing wherein or cause of its reproduction or life cycle produces pathogen protein matter.Pathogenic agent also can be food-borne causal agent.

Bacterial pathogens includes, but is not limited to as bacteriosis originality gram-positive cocci, and it includes, but is not limited to: streptococcus pneumoniae, staphylococcus and suis.Pathogenicity bo Gram-negative coccus includes, but is not limited to: meningococcus and gonococcus.Pathogenicity bo intestines gram negative bacillus includes, but is not limited to: enterobacteria, pseudomonas, actinomycetes, Aitken bacterium (eikenella), pseudoglanders, Salmonellas, Shigellae, influenzae, venereal ulcer, brucella, soil draws bacterium, Yersinia (pasteurella), Streptobacillus moniliformis (streptobacillus moniliformis), spirillum (spirilum), listerisa monocytogenes in mjme (Listeria monocytogenes), erysipelothrix ruhsiopathiae (Erysipelothrix rhusiopathiae), diphtheria, cholera, anthrax, fifth venereal disease (donovanosis) (granuloma inguinale (granuloma inguinale)) and Bartonella (bartonellosis).Pathogenicity bo anaerobic bacterium includes, but is not limited to the bacterium causing tetanus, botulism (botulism), other clostridium disease, pulmonary tuberculosis, leprosy and other mycobacterial diseaseses.Pathogenicity bo spirochetal diseases includes, but is not limited to: syphilis, treponematosis, yaws, pinta and halstern's disease and leptospirosis.Other infection caused by superior pathogenic bacterium and pathogenic epiphyte include, but is not limited to: actinomycosis, nocardiosis, torulosis, blastomycosis, histoplasmosis and coccidioidomycosis, moniliosis, aspergillosis, mucormycosis, sporotrichosis, paracoccidioidomycosis, Podbielniak mycosis, torulopsis, mycetoma, chromomycosis (chromomycosis) and dermatomycosis.Rickettsial infection includes, but is not limited to Rickettsiae and rickettsiosis.The example of mycoplasma and choamydiae infection includes, but is not limited to: mycoplasma pneumonia, lymphogranuloma venereum (lymphogranuloma venereum), psittacosis and perinatal period choamydiae infection.Causative protozoa and worm (helminth) and infectivity eukaryote include, but is not limited to thus: loeschiasis, malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, pneumocystis pneumoniae (Pneumocystis carinii), babesiosis, giardiasis, trichonematosis, filaricide, schistosomicide, nematode, fluke or leech (fluke) and tapeworm (band worm (tapeworm)) infect.Bacterium also includes, but is not limited to listeria bacteria, intestinal bacteria, Campylobacter and salmonella.In some embodiments, intestinal bacteria are colon bacillus 0157.

The example of virus includes, but is not limited to HIV, A type, B-mode and hepatitis C, FIV, slow virus, pestivirus (pestiviruses), west Nile virus, measles, smallpox, cowpox, Ebola virus, coronavirus etc.Other pathogenic agent are also disclosed in U.S. Patent Application Publication case US20080139494, and this case is incorporated herein by reference.

In some embodiments, pathogenic agent is food-borne causal agent.Analyte also may reside on food-borne causal agent.Food-borne causal agent is after edible contaminated food, cause the pathogenic agent of disease (such as viral or bacteroidal).Food itself does not directly cause disease, but it is more specifically present in the consumption of the food-borne causal agent caused on the food of disease.In some embodiments, food-borne causal agent is intestinal bacteria, listeria bacteria, Campylobacter or salmonella.In some embodiments, analyte is selected from food-borne causal agent analyte.Such as, food-borne causal agent analyte can be that (but being not limited to) is selected from intestinal bacteria analyte, listeria bacteria analyte, Campylobacter analyte or salmonella analyte.In some embodiments, analyte is specificity O-antigen.In some embodiments, O-antigen is bacillus coli antigen and/or salmonella O-antigen and may be used for intestinal bacteria and Salmeterol fluticasone propionate.In some embodiments, analyte is flagellin antigen.In some embodiments, analyte is Campylobacter spp flagellin antigen.In some embodiments, analyte is virulence factor gene, such as, from the shiga toxin gene that pathogenic Escherichia coli or Salmonellas increase.In some embodiments, analyte is via amplification method (such as PCR or RT-PCR) amplification and subsequently according to DNA or the RNA sequence that method described herein detects.

As described herein, analyte can be amplified production.Amplified production, as PCR primer (such as double stranded PCR products) can be marked with interaction unit.Can by using by two kinds of interaction unit marks or carrying out the generation of the amplified production marked with unit with the primer of its combination.In some embodiments, analyte will have two kinds of different interaction unit, make bridge joint mixture can be assemblnig and can by detecting signal unit to detect multiple analytes.

As used herein, term " detecting signal unit " means can after testing to determine whether analyte is present in the unit in sample.Detecting signal unit can be can any reagent after testing or composition.In some embodiments, detecting signal unit is connected to capture agent.Therefore, detecting signal unit may be used for detecting the existence of the capture agent being incorporated into its specific binding partner.Capture agent directly can comprise detection reagent, or capture agent can comprise the particle containing detection reagent further.In some embodiments, capture agent and/or particle comprise pigment (color), Radioactive colloidal gold, radioactive labels, fluorescence labels or chemical luminous substrate (substrate).In some embodiments, detecting signal unit comprises near infrared or infrared tags or substrate.In some embodiments, detecting signal unit comprises pigment, Radioactive colloidal gold, radioactive labels, fluorescence labels or chemical luminous substrate.In some embodiments, detecting signal unit comprises nanocrystal, functionalized nanoparticle, upper conversion (up-converting) nanoparticle, cadmium selenide/Cadmium Sulfide merge nanoparticle, quantum dot and can in NIR light spectrum luminous near infrared (NIR) fluorophore or material (as the material of (but being not limited to) such as lanthanon bunch and phthalocyanine, and the photodiode be made up of CuPc, PdPc and PtPc).In some embodiments, capture agent and/or particle are incorporated into detecting signal unit, as (but being not limited to) colloid gold and silver, radioactive labels, fluorescence labels or chemical luminous substrate, near infrared compound (such as substrate, molecule, particle) or infrared compound (such as substrate, molecule, particle), nanoparticle, radioactive nano particle, quantum dot, magnetic particle or enzyme.

Detecting signal unit also can be such as virus particle, latex particle, lipid particle, fluorescent particles, near infrared particle or infrared particle.As used herein, term " fluorescent particles " means particle luminous in fluorescence spectrum.As used herein, term " near infrared particle " means particle luminous near infrared spectrum.As used herein, term " infrared particle " means particle luminous in infrared spectra.In some embodiments, Radioactive colloidal gold has about 20nm, about 30nm or about 40nm or the diameter dimension in the scope of about 20-30nm, about 20-40nm, about 30-40nm or about 35-40nm.In some embodiments, particle comprises metal alloy particle.In some embodiments, metal alloy particle has the diameter of about 10 to about 200nm.The example of metal alloy particle includes, but is not limited to golden metal alloy particle, gold-silver bimetal particle, silver metal alloy particle, copper alloy particles, cadmium-Se particle, palldium alloy particle, platinum alloy particle and plumbous nanoparticle.

As discussed herein, signal detection can be incorporated into the one in analyte.The limiting examples being incorporated into the detecting signal unit of analyte is shown in Fig. 1.Fig. 1 describes in greater detail in herein, shows the detecting signal unit 60 being incorporated into analyte 40 by capture agent 50.But detecting signal unit also can be incorporated into other parts of mixture.Any component that only must exist when multiple analytes is all present in mixture can be all the binding partners of detecting signal unit.Usually (but not exclusively), this component will be the one in analyte, but also can for being incorporated into the capture agent of analyte.By contrast, in some embodiments, detecting signal unit is not just incorporated into the analyte, the solid support thing that are combined with solid support thing or is directly incorporated into the capture agent (if being present on solid support thing) of solid support thing.Such as, in FIG, detecting signal unit will directly not be incorporated into solid support thing 10, capture agent 15 or analyte 20.Be not bound to any particular theory, if detecting signal unit is directly combined with solid support thing 10, capture agent 15 or analyte 20, so method will provide false positive results, and namely signal detects when multiple analytes must not exist.Such as, Fig. 6 description taken in conjunction is in the detecting signal unit of the component of polycomponent bridge-jointing unit.The embodiment of bridge-jointing unit and polycomponent bridge-jointing unit in this article and such as describes with reference to Figure 4 and 5.Fig. 6 illustrates detecting signal unit 60, and wherein its capture agent 50 is incorporated into the component of bridge-jointing unit 30.Bridge-jointing unit 30 comprises particle 34, first capture agent 31, second capture agent 32 and the 3rd capture agent 33.Fig. 6 description taken in conjunction is in the detecting signal unit of the second capture agent 32.Only when two kinds of analytes are all present in mixture, capture agent 32 will be present in mixture.If capture agent 32 does not exist, this means the bridge joint mixture that there is not multiple analytes.Therefore, when multiple analytes is present in mixture, detecting signal unit will only be a part for mixture, avoids false positive results thus.If two kinds of analytes do not exist, so capture agent 32 part that will not be mixture, and therefore, will the binding partners of detecting signal unit be there is not.Therefore, only when multiple analytes exists, detecting signal unit is detectable.Therefore, in some embodiments, detecting signal unit is incorporated into any component only existed when multiple analytes also exists.Other characteristics of polycomponent bridge-jointing unit, characteristic sum constitutional features are also disclosed in herein and are apparent based on the present invention.

The example of the device of the method described at present wherein can be used to be described in such as US Patent No. 8,012,770, U.S. patent application case US13/360, in 528 (submissions on January 27th, 2012), PCT publication WO 2011/044574, each case is incorporated herein by reference in full.But the method described at present can be used with multiple device or form, such as porous plate, array, microarray or in " ELISA " type format.The example of device is also described in herein, but these examples are nonrestrictive.Method described herein also can be combined with cross-flow devices.In cross-flow devices, the different piece of device is in the same plane contrary with vertical current device.The limiting examples of cross-flow devices can be found in US Patent No. 6,485, and 982, US 6,818,455, US6,951,631, US 7,109,042, RE39, in 664 grades, each patent is incorporated to by reference.Cross-flow devices can through adjusting for method described herein, because described method is described for vertical current device.In cross-flow devices, the region of instruction positive or negative result can comprise a kind of capture agent be incorporated in analyte.Bridge-jointing unit may reside in one of transverse flow region or before adding in device and mixes with analyte, and this also can carry out for other devices and solid support thing.Detecting signal unit also can be incorporated in one of transverse flow region.As apparent by the present invention, the type of device or solid support thing is not critical and method can be adjusted based on example described herein and embodiment.

As used herein, term " amplicon " means to be reacted by PCR or the amplified production of other amplified reactions or method amplification, such as nucleic acid molecule.As discussed herein, amplicon can be analyte.Amplicon can be double chain acid molecule.Amplified production can directly or indirectly by use antibody or other capture agent systems (comprise described herein those) detect.Amplified production also can be detected by hybridizing method as described herein in whole or in part.Amplified production also can such as be prepared by RT-PCR or linear amplification.

In some embodiments, amplicon is PCR primer.PCR reaction product (such as amplicon) can make it be detectable by another antibody or antibody sample system through mark, and the BRDU mark of such as (but being not limited to) biotin-avidin/Streptavidin system, system, haptens system, DNA, the intercalating agent of marker DNA, also to may be used for wherein PCR primer through the dNTPS etc. of mark be situation through mark.Analyte can such as (but being not limited to) for nucleic acid (strand or double-strand) and can those identify or detect as described herein by antibody or other capture agent systems.Nucleic acid molecule can be marked with biotin labeling or use the mark of the detectable other types of method described herein.Other examples of mark comprise fluorescent mark.Fluorescent mark can be such as fluorescein (such as luciferin lsothiocyanates (FITC)), rhodamine (such as tetramethylrhodamin (TAMRA)) etc.Amplicon can be produced by using the primer through mark with these marks.Mark can be incorporated in amplicon by amplification program and therefore to become a part for analyte.Mark will be regarded as allos label, because this mark does not find in the native sequences of template being used as amplicon.Can use and be incorporated into mark with the capture agent (such as antibody) contributing to being formed mixture described herein, it makes to detect multiple analytes with single signal.These marks can be used as interaction unit.How Fig. 3 show tags can be used as interaction unit to make it possible to detect with single signal the limiting examples of multiple analytes.

Such as, in one embodiment, PCR reaction is carried out with haptens and/or biotin labeled DNA or RNA primer with analyte nucleic acid sequence homology.Analyte nucleic acid sequence can for (but being not limited to) be from the toxin gene of meat sample and/or lps molecule (such as shiga toxin).But sample can be any sample, and analyte can be the analyte of any other type described herein.PCR reaction can be carried out to produce the multiple analytes with interaction unit.After with primer amplification, method described herein can be used to detect PCR sample.Also with digoxin and/or TAMRA and/or PCR reaction can be carried out with the primer of FITC and TAMRA mark.The amplicon of these marks that can create a difference, amplicon can by using capture agent bridge joint together to make it possible to detect multiple analytes with single signal.The example of this kind of mixture is shown in Fig. 3.

Fig. 3 illustrates to have anti digoxin antibody (namely, capture agent 15), the amplicon of digoxin/TAMRA mark (namely, first analyte 20, first-phase mutual effect unit 21 and second-phase mutual effect unit 22), anti-rhodamine antibody ((namely, bridge-jointing unit 30), FITC/TAMRA mark amplicon (namely, second analyte 40, first-phase mutual effect unit 41 and second-phase mutual effect unit 42) test membrane (that is, solid support thing 10); With the Streptavidin-Au composite (that is, signal generation unit 60 and the 3rd capture agent 50) being incorporated into biotinylated anti-FITC antibody.

Briefly, after carrying out PCR reaction, amplicon can contact with detecting signal unit with solid support thing, bridge-jointing unit.Solid support thing can have the capture agent of the interaction unit be incorporated on the first analyte.Bridge-jointing unit can have or capture agent for being incorporated into the interaction unit on the first and second analytes, makes the interaction unit be incorporated on the first and second analytes make analyte become mixture altogether.Detecting signal unit can be incorporated into the interaction unit be present on one of second analyte.Detecting signal unit can launch detectable signal subsequently, or detecting signal unit can by adding another detection system to detect.Such as, in figure 3, detecting signal unit is the capture agent (such as, antibody) of the interaction unit be incorporated on the second analyte.Detecting signal unit is through biotinylated.The existence of detecting signal unit can be determined subsequently by adding Streptavidin.The mixture that Streptavidin will only be incorporated into two kinds of analytes and all exists.In limiting examples in figure 3, Streptavidin is with colloid gold label, and Radioactive colloidal gold makes it possible to detect.But other marks or detection system may be used for detecting Streptavidin.In the embodiment of vertical current device described herein, test membrane is the solid support thing with capture agent, and pad can comprise the molecule of the combination of the interaction unit of detecting signal unit or detection signal detecting unit and the second analyte.

Fig. 7 illustrates non-limiting workflow procedure, and described program can be used for using amplicon single signal to detect multiple analytes to detect the analyte existence in the sample to which paid close attention to.Analyze foodstuff samples 7000 to determine the presence or absence of pathogenic Escherichia coli.Foodstuff samples 7000 treated (such as, enrichment, cultivation, nucleic acid, purifying, separation, extraction or other similar steps) is with extracting and developing or otherwise obtain the nucleic acid be present in foodstuff samples.The nucleotide sequence be present in treated sample 7001 can increase, and such as (but being not limited to) is by PCR, with specific amplification pathogenic Escherichia coli sequence.The example of these sequences is described in herein.Do not need to use Auele Specific Primer group, because those sequences can be modified based on target sequence to be amplified.As described herein, primer through mark, can produce the amplicon (having the analyte of allos interaction unit) through mark thus.If target sequence is present in foodstuff samples and treated sample, so by generation first analyte 7020 and the second analyte 7040.Display analysis thing has allos interaction unit (7021,7022,7041 and 7042).Analyte can mix with bridge-jointing unit 7030.Mixture will form bridge joint mixture 7100.Can subsequently by making bridge joint mixture contact detect analytes with the solid support thing 7010 comprising capture agent 7015 with the detecting signal unit 7060 comprising capture agent 7050.As discussed herein, the detecting signal unit 7060 comprising capture agent 7050 can be absorbed on film and to allow itself and bridge joint mixture to interact.The solid support thing 7010 comprising capture agent 7015 can for having the test membrane of antibody.These key elements (element) can be incorporated in device as described herein.Although Fig. 7 step display carries out respectively, it also can carry out with different order and some steps can combine.For example, the step making analyte and bridge-jointing unit mix also can contact combined with the detecting signal unit comprising capture agent with making analyte.Can carry out subsequently adding mixture to the detecting step in solid support thing.In some embodiments, analyte, bridge-jointing unit, the signal interaction unit comprising capture agent and the solid support thing that comprises capture agent can simultaneously or almost simultaneously mix and detectable signal detecting unit subsequently.Detecting signal unit only detected when multiple analytes is present in tested sample or (that is, more than negative control) detects more than background level.That is, in the figure 7, if will only at two kinds of analytes and therefore two target sequences are all present in foodstuff samples 7000 time formed mixture 7200.If mixture 7200 will not be formed when one of analyte lacks.The workflow shown in Fig. 7 also can comprise washing step to wash away any non-bond material or component that do not form mixture 7200.Washing step also can be incorporated in any method described herein.

In some embodiments of method described herein, the method detecting multiple analytes with single signal comprises the multiple target nucleic acid sequences increasing and be present in sample.Target sequence can be analyte, or the product of amplification can be analyte.The existence of detection instruction template sequence in primary sample of the sequence (such as, PCR primer) of amplification.

In some embodiments, comprise by the method that single signal detects multiple analytes concurrently and a) use single signal to detect the device of multiple analytes and one or more sample contacts comprising multiple analytes; With the presence or absence of detection signal detecting unit, the presence or absence of the second analyte that described detecting signal unit indicates the first paid close attention to analyte concurrently and pays close attention to.Device can be any device of presence or absence for detect analytes, includes, but is not limited to device described herein.In some embodiments, device comprises: shell, and it comprises: be the import of fluid contact with pad; Afterburning component; Contact the slidably locking component of afterburning component; Contact the transom of afterburning component; The sliding button of contact transom; With the detection membranous system comprising pad, test membrane and absorption component, pad, test membrane and absorption component substantially parallel to each other at least partially, afterburning member contact detects membranous system and can apply to be substantially perpendicular to the pressure detecting membranous system, sliding button moves slidably locking component, and pad comprises the detecting signal unit comprising the 3rd capture agent; Test membrane comprises the first capture agent being fixed on test membrane.

In some embodiments, one or more sample comprises the first paid close attention to analyte, the second analyte paid close attention to and the bridge-jointing unit comprising the second capture agent, the first wherein paid close attention to analyte comprises the first-phase mutual effect unit being incorporated into the first capture agent and the second-phase mutual effect unit being incorporated into bridge-jointing unit, and the second analyte paid close attention to comprises first-phase mutual effect unit in conjunction with bridge-jointing unit and second-phase mutual effect unit.In some embodiments, detecting signal unit comprises the 3rd capture agent, 3rd capture reagent bind is in the component of the mixture of the first-phase mutual effect unit of the second analyte, the second analyte or second-phase mutual effect unit, the first and second analytes, or the component of the bridge-jointing unit only existed when mixture contains the first and second analytes.

In some embodiments, detect the part being included in one or more sample contacted and pad moved after flowing through pad, make the detection be exposed at least partially for detecting signal unit of test membrane thus, thus indicate the presence or absence of multiple analytes with single signal.In some embodiments, by making slidably locking component move pad.In some embodiments, one or more sample contacted with pad before compressed detected membranous system.Method can carry out detecting multiple analytes with multiple sample.Such as, if carry out multiple amplified reaction to produce multiple amplicon (analyte), so multiple amplified reaction is regarded as independent sample separately.In order to detect multiple analytes with single signal, sample must mix.Multiple sample can contact at the forward slip value of contact device or sequentially or simultaneously with device (solid support thing).

In some embodiments, the first and second analytes are amplicon.In some embodiments, the first and second analytes are PCR reaction product.In some embodiments, the first-phase mutual effect unit of the first analyte is digoxigenin labeled.In some embodiments, the second-phase mutual effect unit of the first analyte is rhodamine mark.In some embodiments, the first-phase mutual effect unit of the second analyte is rhodamine mark.In some embodiments, the second-phase mutual effect unit of the second analyte is fluorescein-labelled.In some embodiments, the 3rd capture reagent bind is in the second-phase mutual effect unit of the second analyte.In some embodiments, the 3rd capture agent is biotinylated capture agent.In some embodiments, signal interaction unit is coated with Streptavidin.In some embodiments, signal interaction unit is the Radioactive colloidal gold of Streptavidin coating.In some embodiments, the first and second analytes are nucleic acid amplification product, wherein: the first analyte comprises digoxigenin labeled and rhodamine mark; Second analyte comprise rhodamine mark and fluorescein-labelled; First capture agent is anti-digoxigenin labeled antibody; Second capture agent is anti-rhodamine traget antibody; 3rd capture agent is biotinylated anti-fluorescein labelled antibody; And signal interaction unit is the Radioactive colloidal gold of Streptavidin coating.

If this paper is with used in the whole text, term " (attached) of connection " or " connecting (attachment) " can comprise connected directly or indirectly.Two assemblies be connected to each other directly also are each other in physical contact.Two assemblies connect in succession are to each other connected by intermediate module.Such as, if assembly A is directly connected in assembly C and assembly C is directly connected in assembly B, so assembly A can be connected to assembly B indirectly.Therefore, in this kind of example, assembly A will be called as and will indirectly be connected to assembly B.

Term " capture agent " means can in conjunction with the reagent of target molecule to be detected in sample or analyte.The example of capture agent includes, but is not limited to antibody or its Fab, oligonucleotide and class peptide.Other examples of capture agent include, but is not limited to small molecules or protein, as vitamin H, avidin, Streptavidin, haptens, digoxin, BRDU, strand and double-strandednucleic acid associated proteins or other intercalating agent etc., or identify and catch its molecule.These are the limiting examples of capture agent.Also the capture agent of other types can be used.

As discussed herein, capture agent also can mention such as antibody.Complete antibody (also referred to as immunoglobulin (Ig)) typically is the four polyglycosylated albumen be made up of two weight (H) chains of two of each about 25kDa light (L) chains and Ge Yue 50kDa.The light chain (being called λ and κ) of two types is present in antibody.Depend on the aminoacid sequence of the constant domain of heavy chain, immunoglobulin (Ig) is assigned as five primary categories: A, D, E, G and M, such as, and some in these classifications can be further divided into subclass (isotype), IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.Each light chain holds variable (V) structural domain (VL) and constant (C) structural domain (CL) to form by N.Each heavy chain holds V structural domain (VH), three or four C-structure territories (CH) and a hinge area to form by N.CH structural domain closest to VH is appointed as CH1.By four, VH with VL structural domain is called that the relative conserved sequence region (FR1, FR2, FR3 and FR4) of framework region forms, these regions form the skeleton of three high variable sequence regions (complementary determining region, CDR).CDR contains the interactional most of residue of specificity of responsible antibody or antigen-binding proteins and antigen.CDR is called as CDR1, CDR2 and CDR3.Therefore, the CDR component on heavy chain is called as H1, H2 and H3, and the CDR component on light chain is called as L1, L2 and L3.CDR3 is the largest source of molecular diversity in antibody or antigen-binding proteins binding site.For example, H3 may be as little to two amino-acid residues or more than 26 amino acid.The subunit structure of different classes of immunoglobulin (Ig) and three-dimensional structure are well-known in the art.About the summary of antibody structure, see antibody: laboratory manual (Antibodies:A Laboratory Manual), cold spring harbor laboratory (Cold Spring Harbor Laboratory), the people such as Ha Luo (Harlow) compile, and 1988.Those skilled in the art it should be understood that each subunit structure, and such as CH, VH, CL, VL, CDR and/or FR structure, all comprises active fragments.Such as, active fragments can by the part (that is, Fab) of VH, VL or CDR subunit of conjugated antigen, or is incorporated into and/or activates the part composition of CH subunit of Fc acceptor and/or complement.

The limiting examples of the binding fragment contained in term used herein " antigen-specific antibodies " comprises: (i) Fab fragment, the monovalent fragment be made up of VL, VH, CL and CH1 structural domain; (ii) F (ab') 2 fragment, comprises the bivalent fragment of two the Fab fragments connected by the disulfide bridge bond of hinge area; (iii) the Fd fragment be made up of VH and CH1 structural domain; (iv) the Fv fragment be made up of VL and the VH structural domain of antibody single armed; (v) dAb fragment, it is made up of VH structural domain; (vi) CDR be separated.In addition, although two of Fv fragment structural domain VL and VH are encoded by separate gene, but it can pass through synthetic linker (linker, connexon) restructuring joint, produce wherein VL and VH structural domain pairing with the single protein chain (being called scFv (scFv)) forming monovalent molecule.The most frequently used joint is 15 residue (Gly ₄ser) ₃peptide, but also other joints known in affiliated field.Single-chain antibody is also intended to be embraced in " Fab " of term " antibody or antigen-binding proteins " or antibody.Antibody also can be polyclonal antibody, monoclonal antibody, chimeric antibody, Fab, Fc fragment, single-chain antibody or its any derivative.Capture agent or antibody also can for VHH district, bi-specific antibody, comprise the peptide fragment of antigen binding site or be incorporated into the compound of paid close attention to antigen.The antigen paid close attention to can be the analyte of amplicon or other types.

The routine techniques that these antibody can buy or use those skilled in the art known obtains, and screens the effectiveness of these fragments in the mode identical with complete antibody.Antibody diversity is produced by the germ line genes of Multi-encoding variable domains and multiple somatic events.The restructuring that somatic events comprises variable gene segment and diversity (D) and engages (J) constant gene segment C to prepare complete VH structural domain, and variable and engage constant gene segment C restructuring to prepare complete VL structural domain.Regrouping process, originally as coarse, cause the amino acid loss at V (D) J tie point place or adds.Before antigen-exposed, these diversity mechanism come across in developing B cell.After antigenic stimulation, the antibody gene experience somatic mutation expressed in B cell.Based on the estimated number of germline gene segment, the random restructuring of these sections and random VH-VL pairing, nearly 1.6x10 can be produced ⁷plant different antibodies (basic immunology (Fundamental Immunology), 3rd edition (1993), Borrow (Paul) compiles, crow press (Raven Press), New York (New York), N.Y.).When considering to contribute to other processes of antibody diversity (such as, somatic mutation), thinking and can produce more than 1x10 ¹⁰plant different antibodies (immunoglobulin gene (Immunoglobulin Genes), 2nd edition (1995), the people such as Ji Aoniao (Jonio) compile, academic press (Academic Press), San Diego (San Diego), California (Calif.)).Because many processes participates in producing antibody diversity, independent derivative have the specific monoclonal antibody of same antigen and can not have same acid sequence.

Can by the well-known method of those skilled in the art produce can with antigen described herein, epi-position or the interactional antibody of other molecular specificities or antigen-binding proteins molecule.For example, monoclonal antibody can be produced by producing hybridoma according to currently known methods.Standard method can be used subsequently, the analyses of such as Enzyme Linked Immunoadsorbent Assay (ELISA) and Biacore screen the hybridoma formed by this way, to identify that one or more produce the hybridoma with the molecule paid close attention to or the interactional antibody of compound specificity.

As the replacement scheme of hybridoma preparing secrete monoclonal antibody, can by identifying and be separated the monoclonal antibody of polypeptide of the present invention with separation and combination thus in the immunoglobulin library member of polypeptide with polypeptide of the present invention screening restructuring combination immunoglobin libraries (such as, antibody phage display library).For generation of being that those skilled in the art is well-known with the screening technology of phage display library and commercial reagent box.In addition, especially may be used for produce and the example of the method for screening antibodies or antigen-binding proteins display libraries and reagent can be found in document.

Term " capture agent " also comprises chimeric antibody, such as humanized antibody, and full-length human antibody.In some embodiments, capture agent is goat Chinese People's Anti-Japanese Military and Political College enterobacteria 0157:H7 antibody catalog number (Cat.No.): 70-XG13 (phenanthrene hereby Gerald industry (Fitzgerald Industries)); Colon bacillus 0157: H7 monoclonal antibody catalog number (Cat.No.): 10-E13A (phenanthrene hereby Gerald industry); Colon bacillus 0157: H7 catalog number (Cat.No.): 10C-CR1295M3 (phenanthrene hereby Gerald industry); Colon bacillus 0157: H7 monoclonal antibody catalog number (Cat.No.): 10-E12A (phenanthrene hereby Gerald industry); Or goat anti-mouse IgG catalog number (Cat.No.): ABSE-020 (DCN).Capture agent also can be such as albumin A, Protein G etc.Capture agent also can for combination or specific binding be in the antibody of fluorescent mark (such as, fluorescein or rhodamine), haptens, digoxin etc.Capture agent as Streptavidin can be combined with Radioactive colloidal gold.Streptavidin-Au composite can subsequently for being such as incorporated into biotinylation product, biological example elementization antibody.Limiting examples is found in Fig. 3.Mark shown in Fig. 3 only can use other to arrange for illustration of property object.

Capture agent also can comprise anti-antibody, namely identifies another antibody but to the analyte not specific antibody of tool, such as (but being not limited to) anti-igg, anti-IgM or anti-IgE antibodies.

As used herein, term " concurrently " refers to and simultaneously or almost simultaneously detects multiple analytes.As used herein, " detect the method for multiple analytes concurrently with single signal " or its version refers to the method using the device of single analysis (such as, single hole, single-point, single position on an array) or special purpose to detect with single signal multiple analytes.If different devices, hole or array are used for by same signal detection multiple analytes, so this is not the method detecting multiple analytes with single signal concurrently.Method as detecting multiple analytes concurrently with single signal is wanted for a kind of method, described method only must produce single signal (example of signal is described in herein) at single position (hole on the solid support thing of film or other types, point, line etc.) place, and this informs that user's multiple analytes is present in sample.Such as, by same signal in different hole with indicate single analyte whether to be present in this hole (or the point on array) and the multiple hole of subsequent analysis (or point) to determine whether multiple analytes exists, this is not the method detecting multiple analytes with single signal concurrently.

As used herein, term " single signal " means the signal detection based on single part or method.Such as, if single signal is color redness, the existence so only based on color redness indicates multiple analytes.That is, in this limiting examples, color redness instruction multiple analytes is present in sample.By contrast, if a kind of analyte be by color redness instruction and the second analyte is indicated by color yellow, so use two kinds of colors (that is, signal) be not detect multiple analytes with single signal.Signal is not limited to colorimetric detection.The example of spendable signal is provided herein.

Term " detect (detecting) " or " detecting (detection) " use with most broad sense to comprise the qualitative of target analyte and/or quantitative measurment.

As used herein, term " interaction unit " means analyte or is connected to the part of allos label or tag for analyte, and it is identified by another molecule (such as, capture agent, bridge-jointing unit or detecting signal unit) or combines.Interaction unit can be a part for analyte itself can be maybe allos label or tag.Interaction unit also can be the antibody of discriminance analysis thing or the capture agent of other types.In some embodiments, analyte can comprise more than 1,2,3,4 or 5 interaction unit.In some embodiments, interaction unit is incorporated into be present in analyte itself or the capture agent as another interaction unit on the allos label of a part for analyte.Such as, if analyte is a part for peptide or protein, so the part of protein or peptide itself can be by the interaction unit of capture agent, bridge-jointing unit or detecting signal unit identification.In some embodiments, peptide also can be covalently attached to allos label or tag and the mixture of allos label or tag or peptide and allos label or tag is regarded as interaction unit.Therefore, in some embodiments, analyte comprises first-phase mutual effect unit and/or second-phase mutual effect unit.In some embodiments, one or more interaction unit can be analyte inherently, or one or more interaction unit can pass through some additive methods, as crosslinked, covalently bound by chemical reaction, noncovalent interaction (such as, antibody-antigene, hybridization etc. between the part and another molecule of analyte) add.When interaction unit is the hybridization formation by two kinds of molecules (such as, two nucleotide sequences), make will be regarded as interaction unit by the part of another molecular recognition in hybrid product.Interaction unit also can be added in analyte by amplified reaction.This can produce by using the primer containing interaction unit.Interaction unit also can have detectable signal, but it is not these signals to be detected.

As used herein, the term " allos " about interaction unit means the not natural all groups of analyte, molecule or part.Such as, amplified production can only comprise nucleic acid molecule or nucleotide base.But amplified production can be incorporated into or be connected to allos label, as (but being not limited to) haptens, vitamin H, digoxin, fluorescence molecule (such as, fluorescein or rhodamine) etc.The example of allos interaction unit includes, but is not limited to haptens, vitamin H, nucleic acid molecule, peptide fragment (such as His label, GST label etc.), enzyme, Streptavidin, avidin, fluorescence molecule etc.This inventory is nonrestrictive and can uses any interaction unit.Analyte can with the molecule marker of such as digoxin, rhodamine, fluorescein, DNP, BRDU and subsequently by given molecule being had to the detection of specific capture agent.

According to some embodiments, provide the method detecting multiple analytes.The method of current description may be used for detecting multiple analytes.Unexpected and surprising as a result, can use single signal detect multiple analytes.This has unpredictable consequence, namely only can be detected the presence or absence of multiple analytes by a kind of detection of signal.This with in same reaction, use unlike signal to detect the existence of multiple analytes in sample or needs carry out detecting the independent reaction of multiple analytes and method and formed to the existence detecting multiple analytes and contrast.Namely, embodiment described herein partly provides the method detecting multiple analytes with single signal concurrently, make the existence of multiple analytes in the detection instruction sample of single signal, or there is not existing of multiple analytes in instruction sample in single signal.Embodiment of the present invention provides the method detecting at least 2,3,4 or 5 kind of analyte with single signal concurrently.In some embodiments, method may be used for detecting 2,3,4 or 5 kind of different analyte concurrently with single signal.Although provide the Multi-instance about detection 2 kinds of analytes, can carry out adjusting and amending method about 3,4 or 5 kind of analyte based on the present invention.

As used herein, it is not identical that term " different analyte " means analyte.But different analyte can same title be mentioned, but from different organism or the different strains from same organism.Such as, different organism contains the gene and protein with identical function, and is therefore given same title.But gene or protein are from different sources and are therefore regarded as different analyte.They or can not have different sequence.Different analyte also can mean the analyte from different organism.Such as, there is colibacillary multiple bacterial strain.Not colibacillary all bacterial strains all cause food origin disease.The inventive method may be used for such as detecting the multiple analytes from pathogenic escherichia coli strains, as compared with the analyte detected from avirulence coli strain.Although can mention the analyte of particular type in the present invention, analyte can be the analyte of any type, such as (but being not limited to) analyte classification described herein.

Such as, in some embodiments, the method detecting the first analyte and the second analyte is concurrently provided.In some embodiments, method comprises the solid support thing that makes to comprise the first capture agent and the first analyte, the second analyte, the bridge-jointing unit that comprises the second capture agent contact with the detecting signal unit comprising the 3rd capture agent; With the existence of detection signal detecting unit, detecting signal unit indicates the existence of the first analyte and the second analyte.In some embodiments, the first capture agent is fixed on solid support thing.In some embodiments, the first analyte comprises the first-phase mutual effect unit being incorporated into the first capture agent and the second-phase mutual effect unit being incorporated into bridge-jointing unit; Second analyte comprise in conjunction with bridge-jointing unit first-phase mutual effect unit and be incorporated into the second-phase mutual effect unit of detecting signal unit.Subsequently can detection signal detecting unit.If detecting signal unit detected, so its instruction multiple analytes exists.

Not wishing to be bound by any theory, multiple analytes can be detected concurrently by forming mixture.In some embodiments, mixture comprises solid support thing, the first analyte, the second analyte, bridge-jointing unit and detecting signal unit, and wherein each member of mixture is bonded to each other directly or indirectly.Sample through washing, can retain solid support thing simultaneously, and mixture will only detect when forming mixture.The example of these mixtures is found in Fig. 1-Fig. 3, and it is further described in this article.

In some embodiments, there is provided the method detecting the first analyte and the second analyte concurrently, method comprises: solid support thing and the first paid close attention to analyte, the second analyte paid close attention to, the bridge-jointing unit that comprises the second capture agent are contacted with the detecting signal unit comprising the 3rd capture agent; With the presence or absence of detection signal detecting unit, the presence or absence of the second analyte that detecting signal unit indicates the first paid close attention to analyte concurrently and pays close attention to, wherein: the first capture agent is fixed on solid support thing; The first analyte of paying close attention to comprises the first-phase mutual effect unit being incorporated into the first capture agent and the second-phase mutual effect unit being incorporated into bridge-jointing unit; And the second analyte paid close attention to comprises the first-phase mutual effect unit in conjunction with bridge-jointing unit; Be incorporated into the second analyte, the first-phase mutual effect unit of the second analyte or second-phase mutual effect unit, the mixture of the first and second analytes or the detecting signal unit of the component of bridge-jointing unit that only exists when mixture contains the first and second analytes.

Fig. 1 illustrates can form mixture to detect two kinds of analytes concurrently with single signal.Fig. 1 illustrates the capture agent 15 being fixed on solid support thing 10.Capture agent 15 is incorporated into the first analyte 20.Bridge-jointing unit 30 is incorporated into the first analyte.Bridge-jointing unit is also incorporated into the second analyte 40.Fig. 1 also illustrates the detecting signal unit 60 comprising the capture agent 50 being incorporated into the second analyte 40.Fig. 1 illustrates that this mixture is only formed when each member all exists and can be bonded to each other.Subsequently can detection signal detecting unit.Fig. 3 also display single signal detects the embodiment of two kinds of analytes.Fig. 3 shows specific marker (such as FITC, TAMRA, DIG, vitamin H, Streptavidin etc..), but these marks can be modified according to the present invention.

In some embodiments, method comprises one or more washing step.Washing step may be used for removing unconjugated material.Such as, if solid support thing and the first sample contacts, so solid support thing can through washing to remove any unconjugated material.Be in some embodiments of bead (bead) at solid support thing, bead can wash described bead subsequently with sample contacts.Washing bead is conventional and those skilled in the art is well-known.Can based on used be that the particular solid upholder of key feature changes or selects the method for the solid support thing washing bead or other types usually.

In some embodiments, the sample with the first analyte contacts with solid support thing.In some embodiments, mixture, through washing, makes any material not being incorporated into solid support thing all no longer exist.In some embodiments, solid support thing also from same sample or the different sample contacts comprising the second analyte and/or bridge-jointing unit.Mixture can wash to remove any unconjugated material subsequently again.In some embodiments, the detecting signal unit comprising capture agent is added.Also washing step can be comprised to remove any unconjugated detecting signal unit.Subsequently can detection signal detecting unit, maybe can add another reagent of the existence of detection signal detecting unit.In some embodiments, be simultaneously or almost simultaneously carry out in steps.In the carrying out period of method, washing step can be inserted in due course.

In some embodiments, can before being applied to solid support thing or simultaneously by different analyte or sample mix together.Sample can be such as amplification reaction mixture, its for generation of or attempt produce analyte.In some embodiments, different amplified reaction will be carried out with the multiple analytes that increases.Therefore, before sample or analyte are applied to solid support thing, sample or analyte can be mixed.Sample or analyte also can mix with capture agent and/or bridge-jointing unit with before contacting solid surfaces.

In some embodiments, the first and second interaction unit of the first analyte and the first and second interaction unit of the second analyte are allos interaction unit independently of one another.In some embodiments, the interaction unit of the first analyte and the interaction unit of the second analyte are haptens.In some embodiments, the interaction unit of the first analyte and the second analyte is fluorescein or rhodamine molecule.Therefore, in some embodiments, the first analyte and the second analyte have at least one common interaction unit.The common point of interaction unit will make bridge-jointing unit that two kinds of analytes can be made to become detectable mixture.In some embodiments, the first and second analytes do not have identical interaction unit.In this case, for some embodiments, bridge joint entity by for can make two kinds of analytes each other key connect divalence capture agent (such as, bivalent antibody).Divalence capture agent can be incorporated into the first and second analytes simultaneously.In some embodiments, each interaction unit be present in multiple analytes is different.In some embodiments, some interaction unit are different, but some interaction unit are identical.In some embodiments, analyte comprises haptens interaction unit and vitamin H interaction unit.In some embodiments, the first analyte comprises digoxin interaction unit and rhodamine interaction unit; Second analyte comprises rhodamine interaction unit and FITC interaction unit, and bridge-jointing unit is incorporated into rhodamine interaction unit.Bridge-jointing unit can form the mixture containing the first and second analytes subsequently.This is found in such as Fig. 3.

In some embodiments, multiple analytes is the analyte of same type.Such as, each analyte detected can be peptide.In some embodiments, each analyte is nucleic acid molecule, such as amplified production (such as, amplicon).Analyte can be also any type, includes, but is not limited to analyte described herein.In some embodiments, analyte is different.In some embodiments, the first analyte is amplified production and the second analyte is protein or peptide.Any combination of analyte can be used.

As used herein, term " bridge-jointing unit " or " bridge " mean to make two or more analyte key one or more molecules even in the composite.That is, for example, bridge-jointing unit can be incorporated into the interaction unit on the first analyte and the interaction unit on the second analyte.If only detect two kinds of analytes, then can use a kind of bridge-jointing unit.If detect three kinds of analytes, then can use two kinds of bridge-jointing units.In some embodiments, method uses " n-1 " to plant bridge-jointing unit, and wherein " n " is the number of detected analyte.In some embodiments, use single bridge-jointing unit to detect more than 2 kinds of analytes.The example of bridge-jointing unit includes, but is not limited to immunoglobulin molecules (such as IgM, IgE, IgG, IgA etc.), Streptavidin and comprises multiple capture agent make bridge-jointing unit can be incorporated into molecule more than an interaction unit.In some embodiments, bridge-jointing unit is multivalence capture agent.

In some embodiments, bridge-jointing unit is compound, material or macromolecular mixture.Such as, bridge-jointing unit can comprise the nanoparticle and another antibody that are coated with antibody.In this limiting examples, be coated with antibody nanoparticle can containing be incorporated into analyte or the interaction unit on analyte antibody and also containing the antibody being incorporated into another antibody.Another antibody can be incorporated into different analyte.The interaction of the nanoparticle and another antibody that are coated with antibody can make together with different analyte bridge joints subsequently.The non-limitative illustration of this bridge joint mixture can see in Fig. 4 and Fig. 5, also has description hereinafter.Also other versions of the bridge as mixture can be prepared.Precision architecture and the form of bridge-jointing unit are dispensable, as long as it can by multiple analytes " bridge joint " in the composite.Therefore, bridge can be formed with by analyte bridge joint together by multiple subunit or component.Although bridge-jointing unit can through illustrating and being discussed as bridge joint two kinds of analytes, bridge-jointing unit can be designed to bridge joint more than 2 kinds of analytes, such as 3,4,5 kind or more plant.Therefore, in some embodiments, bridge-jointing unit bridge joint 2,3,4,5 kind or more plant analyte.In some embodiments, bridge-jointing unit bridge joint at least 2,3,4 or 5 kind of analyte.The limiting examples of bridge joint 2 kinds of analytes is only to reach illustrative object and embodiment disclosed herein is not limited to only bridge joint 2 kinds of analytes.

As discussed herein, the inventive method can be applied detect more than 2 kinds of analytes.Such as, the method detecting the first analyte, the second analyte and the 3rd analyte with single signal is concurrently provided.For the detection of other analyte, can adapting method in a similar manner.In some embodiments, method comprises first, second, and third analyte is contacted with detecting signal unit with solid support thing, the first bridge-jointing unit, the second bridge-jointing unit; With the existence of detection signal detecting unit, detecting signal unit single signal indicates the existence of first, second, and third analyte concurrently, and wherein the first analyte comprises first-phase mutual effect unit and second-phase mutual effect unit; Second analyte comprises first-phase mutual effect unit and second-phase mutual effect unit; 3rd analyte comprises first-phase mutual effect unit and second-phase mutual effect unit; Solid support thing comprises the first capture agent of the first-phase mutual effect unit being incorporated into the first analyte; First bridge-jointing unit is incorporated into the second-phase mutual effect unit of the first analyte and the first-phase mutual effect unit of the second analyte; Second bridge-jointing unit is incorporated into the second-phase mutual effect unit of the second analyte mark and the third phase mutual effect unit of the 3rd analyte; And detecting signal unit is incorporated into the second-phase mutual effect unit of the 3rd analyte.Be not bound by any theory, expection can detect analytes concurrently, because first, second, and third analyte forms mixture, wherein mixture comprises solid support thing, the first analyte, the second analyte, the 3rd analyte, the first bridge-jointing unit, the second bridge-jointing unit and detecting signal unit, and wherein each member of mixture directly or indirectly combines each other.

Fig. 2 illustrates the mixture of detection three kinds of analytes that can be formed, and it is similar to example illustrated in fig. 1.Fig. 2 illustrates the solid support thing 10 with the capture agent 15 being incorporated into the first analyte 70.First analyte is incorporated into the first bridge-jointing unit 80.First bridge-jointing unit is incorporated into the second analyte 20, and it is also incorporated into the second bridge-jointing unit 30.Second bridge-jointing unit is also incorporated into the 3rd analyte 40, and it is incorporated into capture agent 50.Capture agent is also connected to detecting signal unit 60.Therefore, Fig. 2 illustrates the limiting examples that how can detect three kinds of analytes with single signal.

Fig. 4 illustrates by the non-limiting bridge joint mixture exceeding a kind of molecule, macromole or material and form.This mixture can be called polycomponent bridge joint mixture.Fig. 4 illustrates the bridge-jointing unit 30 comprising particle 34, first capture agent 31, second capture agent 32 and the 3rd capture agent 33.Bridge-jointing unit 30 can make the first analyte 20 and the second analyte 40 altogether and form key to connect the mixture of the first analyte 20 and the second analyte 40.Fig. 4 illustrates particle 34 (such as, nanoparticle, polystyrene, agarose etc.), particle be coated with directly or by interaction unit be indirectly incorporated into the first analyte 20 the first capture agent 31, be also present in the 3rd capture agent the 33, three capture reagent bind on particle 34 in the second capture agent 32 be combined with the second analyte 40.Can detect this mixture according to method and composition described herein subsequently, this is illustrated in Fig. 5.Fig. 5 shows the bridge joint mixture of Fig. 4, and mixture interacts with the detecting signal unit 60 comprising the capture agent 50 being incorporated into the second analyte 40 with the solid support thing 10 with the capture agent 15 being incorporated into the first analyte 20.As discussed herein, the explanation being incorporated into the detecting signal unit of the second analyte is only for illustration purposes.Detecting signal unit also can in conjunction with other parts of mixture, as long as detecting signal unit is not incorporated into and the interactional analyte of solid support thing or solid support thing itself.Solid support thing 40 can be such as test membrane, the test membrane such as, shown in Fig. 3.Other examples of solid support thing are provided herein.

The invention provides the mixture comprising solid support thing, the first analyte, the second analyte, bridge-jointing unit and detecting signal unit, wherein each member of mixture combines each other directly or indirectly.In some embodiments, solid support thing is incorporated into the first analyte, and bridge-jointing unit is incorporated into the first analyte and the second analyte, and detecting signal unit is incorporated into the second analyte.In some embodiments, solid support thing comprises the first capture agent, first analyte comprises first-phase mutual effect unit and second-phase mutual effect unit, second analyte comprises first-phase mutual effect unit and second-phase mutual effect unit, bridge-jointing unit comprises the capture agent of the first-phase mutual effect unit of one or more second-phase mutual effect unit being incorporated into the first analyte independently and the second analyte, and detecting signal unit comprises the capture agent of the second-phase mutual effect unit being incorporated into the second analyte.

In some embodiments, mixture comprises solid support thing, the first analyte, the second analyte, the 3rd analyte, the first bridge-jointing unit, the second bridge-jointing unit and detecting signal unit, and wherein solid support thing, the first analyte, the second analyte, the 3rd analyte, the first bridge-jointing unit, the second bridge-jointing unit and detecting signal unit combine each other directly or indirectly.In some embodiments, solid support thing is incorporated into the first analyte, first bridge-jointing unit is incorporated into the first analyte and the second analyte, and the second bridge-jointing unit is incorporated into the second analyte and the 3rd analyte, and detecting signal unit is incorporated into the 3rd analyte.In some embodiments, solid support thing comprises the first capture agent, first analyte comprises first-phase mutual effect unit and second-phase mutual effect unit, second analyte comprises first-phase mutual effect unit and second-phase mutual effect unit, 3rd analyte comprises first-phase mutual effect unit and second-phase mutual effect unit, first bridge-jointing unit comprises the capture agent of the first-phase mutual effect unit of one or more second-phase mutual effect unit being incorporated into the first analyte independently and the second analyte, second bridge-jointing unit comprises the capture agent of the first-phase mutual effect unit of one or more second-phase mutual effect unit being incorporated into the second analyte independently and the 3rd analyte, and detecting signal unit comprises the capture agent of the second-phase mutual effect unit being incorporated into the 3rd analyte.

In some embodiments, the method described at present may be used for detecting food-borne causal agent by detecting multiple food-borne causal agent analyte with single signal.Such as, sample can be treated with separate analytes (such as, can be separated or expansion of antigen or toxin, or food-borne causal agent nucleic acid).Multiple analytes (such as, food borne pathogens body protein and/or amplicon) can be detected concurrently by method described herein.Method can provide subsequently the specific larger confidence of testing and avoid false negative result.In some embodiments, the positive findings of the existence of food-borne causal agent is indicated to need detection 2,3 or 4 kind of analyte.The inventive method may be used for by single signal detect analytes concurrently.Single signal provides simpler result to make an explanation, because signal will only can detect when detected multiple analytes is all present in sample.Therefore, if detecting 2 kinds of analytes, so signal will only can detect when two kinds of analytes all exist.In some embodiments, signal only can detect when 3 kinds of analytes exist.Such method can be applied to other detection methods.

In some embodiments, method may be used for detection 3 alanysis thing and tests to provide the positive about food contamination.In some embodiments, the one in analyte is toxin (such as, shiga toxin 1 and/or 2).Toxin itself can be detected, maybe can detect the nucleotide sequence of the generation of coding or control toxin.In some embodiments, the one in analyte is eae gene, and it also can be called virulence factor.Eae gene can be found in or be expressed in such as enterohemorrhagic Escherichia coli.

In some embodiments, the one in analyte is serotype analyte, and it can be the antigen produced by the strain specificity of food-borne causal agent.In some embodiments, serotype analyte is e. coli serotype.In some embodiments, e. coli serotype is O26, O45, O103, O111, O121 and O145.Therefore, in some embodiments, positive test about food source contact scar needs to detect 3 kinds of analytes with single signal, wherein 3 kinds of analytes be shiga toxin (such as, shiga toxin 1 and/or 2), eae gene and be selected from the serotype analyte of e. coli serotype, serotype analyte is O26, O45, O103, O111, O121 and O145.In some embodiments, serotype analyte is by the specific expressed protein of pathogenic strains.In some embodiments, analyte is the nucleotide sequence of coding for antigens.In some embodiments, nucleotide sequence is the fragment of the encoding sequence of antigen.Specific fragment is not crucial and those skilled in the art can determine that sequence or its fragment increase to use ordinary method.As discussed herein, can to increase and alternatively by allos interaction unit labels targets sequence.Can subsequently according to method detect analytes provided herein.

Such as, if test about the positive of virus the nucleotide sequence needing existence two different, so these two nucleotide sequences can use method single signal described herein to detect concurrently, as detected these two nucleotide sequences respectively compare with exceeding a kind of signal.In addition, if the existence of cancer needs to detect the several genes expressed in sample, so can detect gene by using the analyte (such as, by using RT-PCR to carry out amplification gene product) relevant to its expression concurrently according to method single signal described herein.Therefore, the method that describes at present has broad applicability and even can use together with not identical analyte with multiple analyte (target molecule).

In some embodiments, provide the method detecting two or more analyte, comprise and use flowing (vertical or horizontal) device to detect multiple analytes.The example of vertical current device and its using method are provided in US Patent No. 8,012, and 770, US 8,183,059 and U.S. patent application case US13/500,997, US 13/360,528, US 13/445, in 233, each case is incorporated herein by reference in full.Device can be adjusted and detect multiple analytes for using single signal.

Therefore, the embodiment provided herein provides by using vertical current and adopting the device single signal of vertical current to detect the method for multiple analytes.Vertical current allows analyte and/or sample to flow through the layer/film of detection of analytes membranous system." through layer " or " through film " is intended to refer to that sample flows through layer and vertically through described layer.In some embodiments, sample flatly or laterally can not flow through different layers/film.

Following term is combined with the description of various vertical current device.Also illustrate other terms relevant with some vertical current devices or its purposes in the whole text.

Term " pressure actuator " and " power actuator " are used interchangeably and refer to the assembly such as can being implemented pressure by applying power.Power actuator also can be called afterburning component.Example includes, but is not limited to various afterburning component described herein.Other examples include, but is not limited to piston or other solid support structure.Power actuator can improve relative to the position of another assembly, reduce or transverse shifting.Manually or the position that signal processing unit (such as, computer) carrys out control actuator can be passed through.The ability of the position of control actuator may be used for the power (such as pressure) that adjustment puts on another assembly (such as (but being not limited to) detection of analytes membranous system).Put on the power of membranous system by adjustment, the flow rate of sample can be adjusted.Power may be used for making sample keep constant by the flow rate of membranous system, or flow rate can be variable.Flow rate also can stop and sample is rested on the different layers of membranous system.Such as, when sample contacts pad, the flow rate of sample can be zero or close to zero.After being held on pad, increase flow rate by regulating by power actuator applied pressure.Sample can pass through whole membranous system subsequently, and applied force maybe can be regulated to stop (shelving) on another layer of membranous system to allow sample.When samples vertical flow through membranous system time, can manually or by use signal processing unit (such as, computer) accurately adjust power, flow rate can be revised at any time.Flow rate also can adjust based on the number of the film of the absorptivity of the film in membranous system and/or system.Based on absorptivity, (such as, increase or reduce) flow rate can be regulated.

Flow rate can any unit, includes, but is not limited to μ l/ minute or μ l/ second etc. is measured.The flow rate of retention period can be such as 0 μ l/ second, or is less than 1,0.9,0.8,0.7,0.6,0.5,0.4,0.3,0.2 or 0.1 μ l/ second or μ l/ minute.Can manually or by signal processing unit (such as, computer) monitor and carry out adjustment flow rate equally.Except method described herein, can also by known well-known of those skilled in the art and the method for routine adjust and monitor flow rate.In some embodiments, flow rate be about 0 to 1ml/ minute, about 0-10ml/ minute, about 1-9ml/ minute, about 1-8ml/ minute, about 1-7ml/ minute, about 1-6ml/ minute, about 1-5ml/ minute, about 1-4ml/ minute, about 1-3ml/ minute, about 1-2ml/ minute, about 0.5-1.5ml/ minute, about 1-1.5ml/ minute or about 0.5-1ml/ minute.In some embodiments, flow rate is about 1,2,3,4,5,6,7,8,9 or 10ml/ minute.In some embodiments, flow rate is at least 1,2,3,4,5,6,7,8,9 or 10ml/ minute.In some embodiments, flow rate is 1,2,3,4,5,6,7,8,9 or 10ml/ minute.

In some embodiments, comprise shell for the device detecting multiple analytes with single signal, shell comprises the first casing component and second housing component.In some embodiments, the first and second casing components can be constructed as single unit.Shell can comprise import.Import allows sample to introduce in stratographic analysis.In some embodiments, the first casing component comprises import.Import can have the size of the appropriate volume being enough to dispose the solution added in device.In some embodiments, the size of opening (opening) is even as big as disposing about 0.1 to 3ml, about 0.1 to 2.5ml, about 0.5 to 2.0ml, about 0.1 to 1.0ml, about 0.5 to 1.5ml, 0.5 to 1.0ml and 1.0 to 2.0ml.

In some embodiments, shell comprises pad, permeable membrane, test membrane and/or absorption component.In some embodiments, shell comprises detection of analytes membranous system.In some embodiments, detection of analytes membranous system comprises pad, permeable membrane, test membrane and absorption component.In some embodiments, detection of analytes membranous system is not containing permeable membrane.In some embodiments, detection of analytes membranous system comprises in the following order: pad, permeable membrane, test membrane and absorption component.

As used herein, term " pad " refers to and can comprise the film of capture agent or the material of other types.Pad can be rhodia, nitrocellulose, polymeric amide, polycarbonate, glass fibre, film, polyethersulfone, regenerated cellulose (RC), tetrafluoroethylene (PTFE), polyester (such as polyethylene terephthalate), polycarbonate (such as 4, 4-Hydroxy-diphenyi-2, 2'-propane), aluminum oxide, mixed cellulose ester (mixture of such as rhodia and nitrocellulose), nylon (such as polymeric amide, hexa-methylene-diamines and nylon66 fiber), polypropylene, PVDF, high density polyethylene(HDPE) (HDPE)+nucleator " dibenzoic acid aluminium " (DBS) (such as 80u 0.024 HDPE DBS (Porex)) and HDPE.The example of pad also comprises (polyethylene terephthalate), (polyethylene terephthalate), (rhodia and nitrocellulose), water graceful (Whatman) (rhodia and nitrocellulose), the graceful #12-S of water (artificial silk)), (aluminum oxide), (aluminum oxide), Sai Duolisi (Sartorius) (rhodia, such as 5 μm) and the graceful standard 17 (silver soldering glass) of water.Pad also can by with sample or other liquid comes into contact after the material that dissolves make.The dissolving of pad can be carried out, make other layers of system described herein can appear or be exposed to for visual control (such as, detect analytes) or for spectrometer inspection (such as, by spectrometer analyte).

In some embodiments, pad or test membrane comprise capture agent.In some embodiments, pad or test membrane contacted with capture agent and make it dry subsequently, subsequently in vertical current device.Pad or test membrane also can comprise other compositions to preserve capture agent, and it can at room temperature or refrigerated or stable storage under freezing temp.In some embodiments, pad or test membrane soaked with damping fluid before using capture agent.In some embodiments, damping fluid is the blocking-up damping fluid for preventing non-specific binding.In some embodiments, damping fluid comprises borate, BSA, PVP40 and/or Tween-100 or its any mixture.In some embodiments, damping fluid is 10mM borate, 3%BSA, 1%PVP40 and 0.25%Tween-100.In some embodiments, capture agent is in the solution comprising trehalose and sucrose, be applied to pad or film.In some embodiments, capture agent be applied in the solution comprising trehalose, sucrose and phosphoric acid salt and/or BSA pad, film or both.In some embodiments, capture agent uses in the solution of 5% trehalose, 20% sucrose, 10mM phosphoric acid salt and 1%BSA.In some embodiments, test membrane comprises the capture agent be incorporated into through marking amplicon.In some embodiments, capture agent is the antibody identifying or be incorporated into digoxin, fluorescein (such as FITC), rhodamine (TAMRA) etc.

In some embodiments, pad comprises Streptavidin.Streptavidin also can as described herein further through mark.In some embodiments, Streptavidin is the capture agent of the biotinylated antibody be incorporated into for detecting multiple analytes with single signal.

In some embodiments, removable member contact pad first surface and adhesion component (adhesive member) contacts the second surface of pad.

In some embodiments, device comprises adhesion component.Adhesion component can comprise permission sample and flow through pad and the adhesion means inlet of Contact test film.In some embodiments, adhesion means inlet and removable member entrance have same size or shape.In some embodiments, adhesion means inlet and removable member entrance have different size or shape.In some embodiments, the entrance in adhesion component has same shape, but has different area.The entrance with different area will be regarded as having different size.Adhesion component can be made up of any material being applicable to a kind of component or film to adhere to another component or film.In some embodiments, adhesion component impenetrable liquid.In some embodiments, adhesion member contact removable member.

In some embodiments of device, permeable membrane is connected to or adheres to test membrane.In some embodiments, permeable membrane is laminated on test membrane.Permeable membrane can for the film allowing sample (such as fluid sample) to flow through any material of test membrane.The example of test membrane includes, but is not limited to soluble cotton, Mierocrystalline cellulose, glass fibre, polyester, polypropylene, nylon etc.In some embodiments, permeable membrane comprises opening.Opening can exist allow range estimation or detect test membrane.In some embodiments, the opening in permeable membrane has same size with the import in shell substantially.It is graceful etc. that the example of permeable membrane includes, but is not limited to Protran BA83, water.

As discussed herein, an example of solid support thing is test membrane.If this paper is with used in the whole text, " test membrane " refers to the film of the binding partners wherein detecting capture agent.Test membrane includes, but is not limited to nitrocellulose membrane, nylon membrane, polyvinylidene fluoride film, poly (ether sulfone) film etc.Test membrane can be can by those skilled in the art for detecting any material of the existence of the binding partners (analyte such as, through marking, antigen or epi-position) of capture agent.Test membrane also can comprise capture agent.In some embodiments, test membrane contacts with capture agent and makes capture agent dry and adhere to test membrane.The example of test membrane includes, but is not limited to that Protran BA83, water are graceful, Opitran BA-SA83 and 0.22 μm white light facial mask (Mi Libo (Millipore) production code member SA3J036107).Test membrane also can comprise the nanoparticle matrix with capture reagent bind.Nanocrystal can be arranged to have such as (but being not limited to) based on the 2D thin slice of the material of the particle of carbon, gold or metal alloy particle, copolymer matrix and single dispersing semi-conductor, magnetic, metal and ferroelectric nano crystal and 3D matrix.Test membrane can comprise multiple capture agent.In some embodiments, test membrane comprises 1,2,3,4,5,6,7,8,9 or 10 kind of capture agent.In some embodiments, test membrane comprises multiple region respectively with different capture agent.In some embodiments, multiple region not exclusively overlaps each other or unanimously.

In some embodiments, device or shell also comprise absorption component.Absorption component also can be called " core pad " or " wicking pad ".The sample administration that is absorbed in absorption component flows through the fluid of device and is provided in the wicking-power that sample administration contributes to sample flow when device when device." absorption component " is intended to refer to have and draws (core) from the surface with material and keep the material of the ability of solution.Use the combination of the material increasing or reduce specific absorption that Quality control also can be allowed to move.

Absorption component can be any material that sample can be promoted to flow through pad and flow to test membrane.The example of absorption component includes, but is not limited to Mierocrystalline cellulose, superabsorbent polymers, glass fiber mats (such as C083 (Mi Libo (Millipore))) etc.In some embodiments, shell comprises multiple (such as, more than 2 or 2) absorption component.In some embodiments, shell comprises 2,3,4 or 5 absorption components.In some embodiments, device comprises an absorption component.In some embodiments, absorption component comprises one or more film (being up to 10 indivedual films), and each film can be same material or differing materials.In some embodiments, device is only made up of 1 film as absorption component.

In some embodiments, device comprises afterburning component.The example of afterburning component is described in hereinafter and is found in figure.These examples are nonrestrictive and can use other forms of afterburning component.In some embodiments, other assemblies that afterburning component may be used for applying pressure or compressing detection of analytes membranous system make to abut against each other.In some embodiments, afterburning component can by including, but is not limited to plastics or stainless any material generation.As shown in Figure 23, fixture can be used as afterburning component.Stainless steel can be laser cutting, makes it to be used as fixture.The limiting examples of these fixtures is found in Figure 23.Afterburning component is used for applying pressure to membranous system.Afterburning component is not limited to fixture, but can be any shape (figure see about limiting examples), it can apply pressure to the piston spline structure at membranous system (such as, nanoparticle matrix) and the key position place being positioned at subassembly.In some embodiments, afterburning component is piston.Other assemblies that afterburning component may be used for applying pressure or compression detection of analytes membranous system make to abut against each other.In some embodiments, afterburning component can comprise axle and head.Afterburning component can have mushroom-shaped shape, and wherein head is wider than axle.In some embodiments, head is narrower than axle.The afterburning component comprising head and axle can be that single unit or multiple parts that can form afterburning component by contacting with each other are made.For example, head can be a unit that can be separated with axle.When assembling, head and axle contact with each other to make afterburning component.In another example, head and axle be a bonding element and together with manufacture, but not assembling forms the individual components of afterburning component after a while.Afterburning component allows device to work together with vertical current, as with depend on transverse flow and compare.

In some embodiments, the surface of afterburning member contact absorption component.In some embodiments, the afterburning surface of member contact absorption component and the surface of layer can be removed.In some embodiments, afterburning component is from compressive films detection system above and below film detection system.Such as, in some embodiments, afterburning component can be clipped in the middle of all layers of film detection system.In some embodiments, afterburning component is connected to bracing member.

In some embodiments, device comprises removable member, pad and adhesion component in the following order.

Device can also comprise bracing member.In some embodiments, the surface of bracing member contact absorption component.Bracing member can also have bracing member entrance.Entrance can have same size and/or shape with removable member and/or the entrance of adhering in component.In some embodiments, bracing member comprises the entrance with removable member and/or the entrance of adhering in component with different size and/or shape.By any material, plastics can be included, but is not limited to and make bracing member.In some embodiments, second housing component is used as bracing member.

Device described herein may be used in the analysis of the existence of the binding partners detecting capture agent.These analyses as shown herely can detect multiple analytes for the detection for single signal.Such as, apparatus of the present invention can be used to carry out detectable antigens by antibody.Term " vertical current " uses in the whole text.Term " vertical current " refers to that sample flow passes through to be present in the direction of different film in device and component.Vertical current refers to the sample flowing through film (such as, top-to-bottom), and as compared with transverse flow, transverse flow refers to the sample flowing through (such as, side is to side) film, pad or absorption component.In cross-flow devices, film and pad are flatly positioned at position adjacent one another are, substantially at grade.In vertical current device, each film or pad substantially parallel or fully parallel to each other and substantially different in occupying device space planes.Film and pad can at it compressed or under stress place time occupy similar plane.In some embodiments, each component, film or pad at least partially in top of each other stratification.In some embodiments, each layer substantially parallel to each other at least partially of component, film or pad.In some embodiments, being at least partially in the space plane different from other layer of each layer.

In order to make vertical current effectively occur, in some embodiments and when it is present, pad, permeable membrane, test membrane and absorption component are substantially parallel to each other.In some embodiments, pad, permeable membrane, test membrane and absorption component are present in different space planes.In some embodiments, shell also comprises hydrophobic membrane, and hydrophobic membrane can slow down or stop the vertical current of sample.Hydrophobic membrane can contact with test membrane, and this will make sample stop or be shelved on test membrane.Stop can allow susceptibility and the detection of increase.Vertical current is regulated by the pressure putting on film, pad and/or component.In some embodiments, pressure is applied perpendicular to test membrane and/or pad.In some embodiments, can apply pressure makes pad compressed against shell.Compression against shell can make pad directly contact with shell, O type ring or hoop or be contacted by intermediate, pad and test membrane is abut against each other compressed.

Afterburning component can apply the pressure being substantially perpendicular to test membrane.Be not bound to any particular theory, pressure promotes vertical current.Pressure makes each layer of membrane stack contact with another layer.Pressure also can solution flow divided by stopping, and make test sample to stop or to be shelved on test membrane, this measure can allow larger susceptibility.Pressure can apply to allow vertical current to continue sample is flowed in one or more absorption component subsequently again.Afterburning component can apply pressure, makes pad contact a part (such as, first or second housing component or layer can be removed) for shell.In some embodiments, when pad is not under afterburning component applied pressure, pad contact shell, but executing on stressed afterburning component, pad is compressed against a part for shell.

In some embodiments, the circumference of pad contact import.Import can also comprise hoop or other similar characteristics, such as O type ring.In some embodiments, the circumference of pad contact hoop and/or O type ring.In some embodiments, pad can be compressed against the circumference of import, and import can comprise hoop and/or O type ring in some embodiments.

" can be compressed against the circumference of import " refer to film or pad (such as, pad) directly contact with the circumference of import compressed or against another layer contacted with the circumference of import or material (such as, film) compressed.

In some embodiments, pad is not that direct physical contacts with shell, but is fluid contact with shell." fluid contact " is applied to device if mean sample by import or other openings, and so fluid will contact pad.In some embodiments, pad can be separated with shell by another film (such as permeable membrane), and wherein another film and shell are that direct physical contacts or is that direct physical contacts with hoop or O type ring.When sample administration is in device, first fluid can contact another film and contact pad subsequently.This is just in time an example of the pad being fluid contact with shell.There are other embodiments numerous, wherein pad is not that direct physical contacts with shell, hoop or O type ring, but is fluid contact with shell.

Afterburning component can apply any pressure being enough to promote vertical current by different rete.In some embodiments, the layer (such as, pad, permeable membrane, test membrane and absorption component) of device is compressed under the power being selected from about 5lbf to 100lbf, about 5lbf to 50lbf, about 10lbf to 401bf, about 15lbf to 40lbf, about 15lbf to 25lbf or about 30lbf to 40lbf.In some embodiments, the layer (such as, pad, permeable membrane, test membrane and absorption component) of device is compressed under the power being selected from about 1lbf to 100lbf, about 1lbf to 50lbf, about 1lbf to 51bf, about 1lbf to 10lbf, about 1lbf to 15lbf, about 1lbf to 20lbf, about 1lbf to 30lbf or about 1lbf to 25lbf.If hydrophobic membrane or impermeable membrane are present in device, power can also compress hydrophobic membrane or impermeable membrane.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, the first surface of afterburning member contact absorption component.In some embodiments, pad Contact test film.In some embodiments, the first surface contact permeable membrane of test membrane.In some embodiments, the second surface of the second surface contact absorption pad of test membrane.In some embodiments, device comprises hydrophobic membrane and such as, the second surface of hydrophobic membrane Contact test film.In some embodiments, the first surface of hydrophobic membrane contact absorption pad.In some embodiments, pad contact adhesion component.In some embodiments, test membrane contact adhesion component.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, the first surface of pad contacts shell and the first surface of the second surface of pad contact permeable membrane, the wherein first surface of the second surface Contact test film of permeable membrane, the wherein first surface of the second surface contact absorption pad of test membrane, wherein the second surface of absorption pad contacts afterburning component.In some embodiments, the circumference of the import of the first surface contact shell of pad.In some embodiments, the first surface contact hoop of pad or the circumference of O type ring.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, the first surface of first surface contact shell component and the contact of the second surface of pad is adhered of pad, wherein to adhere the first surface of second surface Contact test film of component, the wherein first surface of the second surface contact absorption pad of test membrane, wherein the second surface contact bracing member of absorption pad.In some embodiments, the circumference of the first surface contact entrance of pad.In some embodiments, the first surface contact hoop of pad or the circumference of O type ring.

Device can also comprise transom.In some embodiments, transom is flexible or is made up of flexible materials.In some embodiments, transom is fixing or is made up of non-flexible material.Fixing transom can be such as hinge etc., and it can the pad of such as contact system or another layer or film and can mediate its displacement.Fixing transom, the hinge that such as (but being not limited to) is fixing or other compressible materials that hinge-like works, can recover shape or size when compressing release.Transom can make pad be shifted.Although transom can moreover be only plastics and flexible, its curved characteristic cannot be used for the function of device.

Flexible materials can be such as elasticity or elastomer material.Transom can such as be connected to pad and/or hydrophobic membrane.Transom can also be connected to any film or the component of device.The example of transom includes, but is not limited to elastomeric band, string rubber, spring etc.In some embodiments, transom can be made up of shape-memory material.In some embodiments, transom makes likely to be delayed at moving locking member and mobile pad or the film of any other type be connected with transom or between padding.In some embodiments, the movement of pad or film can not occur with mobile sliding button or locking component simultaneously.May be used for detecting in some embodiments of the device of multiple analytes with single signal, and be not bound to any particular theory, when sliding button or locking component move, energy gather in transom and this energy for straining the pad or film that are connected to transom after pressure discharges.In some embodiments, before moving or removing pad, locking component moves (that is, afterburning component no longer contacts locking component) in some embodiments away from afterburning component, once remove the compression or pressure that are applied by afterburning component completely, namely pad moves.

Transom can also be connected to sliding button or locking component.Transom can by any mode, and such as tackiness agent, bail, tying etc. are connected to other assemblies.In some embodiments, film or pad have recess in film or pad, and it allows transom to be connected to film or pad.Limiting examples is found in Fig. 9.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, shell comprises locking component.Locking component can be can the slidably locking component of movement in device.Locking component to may be used for afterburning member lock, in a position, the power produced by afterburning component being on the different layers maintained.Locking component be such as by afterburning member lock in a position, pressure cannot be removed, until locking component moves to allow afterburning component to change position (that is, reducing).Locking component such as can be arranged on the below of the head of afterburning component, this position that will afterburning component made to remain on raising.Locking component can also, through location, make it afterburning component be remained on specific position (such as, improve or reduce).Locking component can be made by any material including, but is not limited to plastics etc.Locking component can such as directly or indirectly through preventing the afterburning component of another component touch of afterburning component relief pressure.In some embodiments, locking component contacts afterburning component to compress pad.

Locking component can also contact transom, makes the movement of locking component by mobile link component, any other film (such as, pad, hydrophobic membrane, test membrane or absorption component) being connected to transom or other assemblies.Such as, if locking component moves the pressure removing afterburning component, afterburning component is made to change position (such as thus, from the position mentioned to the position reduced), so the movement of locking component also will make transom be out of shape/by energy accumulation in transom, therefore once pressure fully reduces, locking component moveable diaphragm or pad.When pad is connected to transom and locking component moves, once pressure fully reduces, this measure is also by mobile pad.In some embodiments, pressure is completely removed.Pad can such as move, and it is removed from device.In some embodiments, pad moves to appear test membrane by import.The amount of the test membrane appeared will depend on the type of the detection of use.Naked eyes are detected, can need to appear more test membrane in import.Non-naked eyes (such as, fluorescence, near infrared, infrared, radioactivity or chemoluminescence) are detected, can need to appear less or more test membrane.In some embodiments, pad moves, and makes no longer can be seen by import or pad be detected.In some embodiments, the movement of pad can produce another opening except import with range estimation or detect test membrane.In some embodiments, pad dissolves with range estimation or detects test membrane (such as, by single signal detect analytes or multiple analytes).Pad can be made up of soluble material, and make when pad contacts with sample or another solution, pad partly or completely dissolves.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, transom is also connected to impermeable membrane or hydrophobic membrane.When transom moves, mobile also by movement or remove impermeable membrane or hydrophobic membrane.As discussed herein, existence that is not saturating or hydrophobic membrane can by slowing down or stopping vertical current making test sample stop or be shelved on test membrane.When impermeable membrane or hydrophobic membrane move or remove, be connected to transom by making it or by other means, vertical current is no longer prevented from or suppresses.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, shell comprises sliding button.Sliding button also can be described as sliding component.Sliding button can pass crosswise internal surface and the outside surface of (cross) shell.In some embodiments, sliding button or sliding component are projected into the outside surface of shell.In some embodiments, sliding button is directly or indirectly connected to locking component.When sliding button connects (directly or indirectly) in locking component, the movement also moving locking member of sliding button.Transom can be connected to sliding button in some embodiments.In some embodiments, transom is connected to sliding button and locking component.Sliding button and locking component also can be configured to single unit.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, any one or more entrances (inlet) all comprise and are selected from about 0.2 to about 20cm ²the opening (opening) of scope.In some embodiments, any one or more entrances are the diameter of about 1 to about 2cm.In some embodiments, any one or more entrances are the diameter of about 1 or about 1.5cm.In some embodiments, any one or more entrances be about 1, the diameter of about 2, about 3, about 4 or about 5cm.In some embodiments, exist more than an entrance, entrance can be different size or same size.The size of each entrance is independent of one another.In some embodiments of device described herein and system, device or system comprise 1,2,3,4 or 5 entrance.In some embodiments of device described herein and system, device or system comprise at least 1,2,3,4 or 5 entrance.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, import comprises and is selected from about 0.2-20cm ²the opening of scope.In some embodiments, import is the diameter of about 1 to about 2cm.In some embodiments, import is the diameter of about 1 or about 1.5cm.In some embodiments, import be about 1, the diameter of about 2, about 3, about 4 or about 5cm.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, the device for detect analytes comprises the first component and second component.In some embodiments, the first component and second component contact with each other.In some embodiments, the first component comprises one or more entrance.In some embodiments, between the first and second components be detection of analytes membranous system.In some embodiments, the detection of analytes membranous system between the first and second components comprises pad, adhesion component, test membrane and absorption component.In some embodiments, detection of analytes membranous system comprises in the following order: pad, adhesion component, test membrane and absorption component.As discussed herein, in some embodiments, substantially parallel to each other at least partially in pad, test membrane and absorption component each.In some embodiments, being at least partially in different spaces plane in pad, test membrane and absorption component each.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, detection of analytes membranous system is compressed between (such as, afterburning component) first and second components.In some embodiments, detection of analytes membranous system is compressed between the plane formed by the first component and the plane formed by second component, and the plane wherein formed by the first and second components is substantially parallel to each other and parallel with detection of analytes membranous system.In some embodiments, plane is parallel to each other and parallel with detection of analytes membranous system.In some embodiments, the first and second components compressing detection of analytes membranous system are afterburning components.Such as, afterburning component can be called and comprises the first and second components to produce the power of compression detection of analytes membranous system.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, the first and second member parallel are connected to each other in the edge of first component at the edge of second component.In some embodiments, the first and second components are by the connection such as spring, hinge.The mode that first and second components connect is unrestricted and can be by making any structure that analyte membranous system can be compressed between the first and second components.In some embodiments, the first and second components are adjacent one another are and form fixture.The example of fixture (such as, afterburning component) shows (such as Figure 16) in subject application in the whole text.Fixture can, such as from metal or make the first component have the material cutting of flexible any type, make detection of analytes membranous system can insert between the first and second components.In some embodiments, the first component is removable.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, the first component connects with pad or contacts, wherein the moving or remove mobile pad or remove pad from device of the first component.In some embodiments, pad is removable.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, pad removes from the device comprising the first and second components by only removing pad.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, pad comprises trim plate (tab).Trim plate may be used for removing or promote to remove pad.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, device described herein is positioned in container.In some embodiments, container is capsule or bag.In some embodiments, container comprises entrance.In some embodiments, container comprises removable or movable member or layer, and component or layer, mobile or expose entrance when removing, allow sample administration in detection of analytes membranous system.Removable or movable member or layer example includes, but is not limited to wing flap (flap) or trim plate.Wing flap or trim plate are such as shown in Figure 18 and Figure 19.In some embodiments, layer can be removed or displaceable layers also can be used as the sealing (seal) of container.Sealing can protect pad and/or detection of analytes membranous system.

In some embodiments of device described herein and system, removable or displaceable layers contacts with pad or connects.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, device for detect analytes comprises the first external member (outer member) and comprises the second external member of the first internals (inner member) and the second internals, and wherein the first internals and the second internals contact with each other.In some embodiments, the first external member comprises entrance.In some embodiments, the first internals comprises entrance.In some embodiments, the first external member and the first internals comprise entrance.In some embodiments, between the first and second internals, be detection of analytes membranous system.In some embodiments, device comprises pad.In some embodiments, device lacks pad.In some embodiments, detection of analytes membranous system comprises test membrane and absorption component and comprises pad alternatively.In some embodiments, detection of analytes membranous system comprises test membrane and absorption component in the following order.In some embodiments, substantially parallel to each other at least partially in optional pad, test membrane and absorption component each.In some embodiments, as discussed above, detection of analytes membranous system is compressed between the first internals and the second internals.In some embodiments, device and/or system comprise component of adhering as described herein.In some embodiments, device comprises filter membrane.In some embodiments, filter membrane can in detection of analytes membranous system.In some embodiments, the first surface of filter membrane contacts the surface of the first internals and another film of the second surface contact analysis quality testing of filter membrane survey membranous system or component.In some embodiments, the surface of the second surface Contact test film of filter membrane.Filter membrane can be any material as described herein.Such as, in some embodiments, filter membrane can for can be used as the same material of pad, test membrane, absorption component etc.In some embodiments, filter membrane is glass fiber mats.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, when pad is not present in device or system, binding substances is in liquid form or in the material forms supply be dissolvable in water in liquid (such as, water, buffered soln, physiological saline etc.).Binding substances can be supplied and be provided by device described herein or system in autonomous container (such as, managing).Be supply seasonable in a reservoir at binding substances, binding substances was hatched together with sample (incubate) before detection of analytes membranous system in sample administration.Sample can produce by any method and/or as described herein.Such as, piece of meat can through clean or wiping and produce test sample.Test sample can hatch together with binding substances subsequently or contact to produce test sample-binding substances mixture.This mixture can use device as described herein and/or system to execute subsequently and be applied to detection of analytes membranous system as described herein.In some embodiments, test sample-binding substances mixture and be directly applied to test membrane.In some embodiments, testing sample-binding substances mixture is after filtration or through another film before Contact test film.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, detection of analytes membranous system is compressed between the first and second internals.In some embodiments, detection of analytes membranous system is compressed between the plane formed by the first internals and the plane formed by the second internals, wherein substantially parallel to each other with the plane that the second internals is formed and parallel with detection of analytes membranous system by the first internals.In some embodiments, plane is parallel to each other and parallel with detection of analytes membranous system.In some embodiments, plane is arranged essentially parallel to the first and second external members.

In this paper some embodiments with device described in the whole text, pad is not compress by the first and second internals or by afterburning component described herein.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, the first external member comprises removable or removable trim plate.In some embodiments, pad is connected to described first external member.In some embodiments, pad is connected to removable or removable trim plate.In some embodiments, the first external member and the second external member form container and container encapsulation first and inner second component.In some embodiments, container be capsule, bag (such as, can the surrounding detection of analytes membranous system and be compressed in the container between the first and second internals of sealable (such as slide fastener, adhesion etc.) or any other type.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, container comprises removable or removable trim plate.Removable or removable trim plate can for any shape and can for fully removable or through removing to the degree exposing entrance.In some embodiments, trim plate is mobile or remove when removing or mobile pad.The removable such as sufficient distance of pad, makes it possible to the result analyzing (such as, estimating) test membrane.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, the first surface of pad contacts with the first external member and the second surface of pad contacts with the first internals.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, the edge that the first and second internals edges are parallel to first internals at the edge of the second internals is connected to each other.In some embodiments, the first and second internals are connected by spring, hinge etc.The mode that first and second internals connect is unrestricted and can be undertaken by making any structure that analyte membranous system can be compressed between the first and second components.In some embodiments, the first and second internals are adjacent one another are and form such as fixture.The example of fixture be shown in subject application in the whole text in.Fixture can, such as from metal or make the first internals have the material cutting of flexible any type, make detection of analytes membranous system can insert between the first and second components.In some embodiments, the first internals is removable.

As discussed herein, device and system can comprise removable or displaceable layers (such as, trim plate).Can by manual force, such as (but being not limited to) is peeled off (pealing) or is torn to remove or mobile removable or displaceable layers.Also can be removed by mechanical force or mobile removable or displaceable layers.Mode that is removable or displaceable layers movement can be any mode.Example that is removable or displaceable layers includes, but is not limited to trim plate, wing flap etc.As discussed herein, this wing flap or trim plate can be used as sealing etc.

As discussed herein, pad can comprise analyte specificity capture agent.In some embodiments, pad comprises multiple analytes specificity capture agent.In some embodiments, pad comprises 1,2,3,4 or 5 kind of analyte specificity capture agent.This analyte can be can by any molecule of capture agent specific recognition.The example of analyte comprises polynucleotide molecule (such as DNA, RNA, siRNA, antisense oligonucleotide), peptide, protein, sugar, polysaccharide, carbohydrate etc.Antigen also can refer to the different epi-positions be present on same protein or polypeptide.Analyte also can refer to the antigen from pathogenicity bo or avirulence organism.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, device can individually, in pairs or in various configurations casing.Shell can for watertight to prevent seepage and can by multiple inert material, such as polymer materials manufacture.In some embodiments, entrance can have the volume treating sample or the reagent used together with the present invention being enough to hold any aequum.

Because the film of device, component or pad are chemically inert in some embodiments, so it may for solvent transport, to make specificity combinating reagent fix required any reaction site place activated at needs.Multiple method may be needed to make Immobilized reagents according to the specified chemical character of reagent.In general, when medium be soluble cotton or mixing soluble cotton ester time, do not need special chemical key connect make Immobilized reagents.Multiple technologies may be used for other materials and reagent, and it comprises functionalized with the material of such as carbonyl dimidazoles, glutaraldehyde or succinic acid or by the material processing of such as cyanogen bromide.Other suitable reactions comprise uses Schiff's base and hydroborate process to reduce aldehyde, carbonyl and amino.DNA, RNA and some antigen can for solvent transport by curing and immobilization on chromatographic material.Cure and can carry out at the temperature within the scope of about 120 DEG C between about 60 DEG C, continue the time of change between about 5 minutes to about 12 hours, and in some embodiments, at about 80 DEG C, carry out about 2 hours.

Embodiment described herein also providing package contains the system of device described herein and buffer container.System may be used for detecting multiple analytes with single signal.Buffer container can for being applied to any damping fluid of device with tested sample mix and subsequently.Such as, sample can obtain from source and sample can mix with damping fluid.Damping fluid can be the damping fluid that the pH of the dissolving damping fluid (lysis buffer, lysis buffer) or maintenance sample that will dissolve (cracking, lyse) cell makes it possible to suitably carry out analyzing.Buffer container can be any shape and can be included in the outside or inside of the shell of device.

In some embodiments, providing package is containing the system of sample divider.Sample divider can for can obtain sample from source and any material allowing sample tested.Such as, sample divider can be swab, such as cotton swab.In some embodiments, sample divider is inoculator.In some embodiments, shell comprise sample divider and a part for sample divider in the inside of shell.In some embodiments, sample divider is partly in the outside of shell and partly in the inside of shell.In some embodiments, sample divider is fully in the outside of shell.

Also provide the test kit detecting multiple analytes with single signal, wherein test kit comprises device described herein.Test kit can comprise device as described herein, sample divider, buffer container, specification sheets, positive control, negative control thing or its any combination.About test kit, positive control is the sample of known one or more analytes containing detecting with the device be present in test kit.By contrast, negative control thing will not contain the analyte that can be detected by test kit.Such as, negative control thing can confirm that when being combined with anti-antibody device suitably works.

Damping fluid can also be included in the present invention.The example of damping fluid includes, but is not limited to 1X PBS (10mM phosphoric acid salt, 137mM sodium-chlor, 2.7mM Repone K), lavation buffer solution (such as, 10mM sodium phosphate, 150mM NaCl, 0.5% tween 20,0.05% sodiumazide), film damping fluid (such as, 10mM sodium phosphate, 0.1% sucrose, 0.1%BSA, 0.2%PVP-40 pH7.21, with 0.2 μm of metre filter), polyclone binding substances block damping fluid (such as, 50mM borate, 10%BSA, pH8.93); Polyclone binding substances thinner (such as, 50mM borate, 1%BSA, pH9.09) or blocking-up damping fluid (such as, 10mM sodium phosphate, 0.1% sucrose, 0.025%Silwet pH7.42; 10mM sodium phosphate, 1% sucrose, 1% trehalose, 0.01%BSA, 0.025% tween 20; 0.05% sodiumazide, 0.025%Silwet pH7.4; 10mM sodium phosphate, 0.1% sucrose, 0.1%BSA, 0.2%PVP-40 pH7.21).Damping fluid can also block damping fluid (such as, 10%BSA (pH7.4) in deionized water or 1%BSA in deionized water (pH7.4)) for (but being not limited to); 10mM borate, 3%BSA, 1%PVP40 and 0.25% tween-100 etc.

Pad and test membrane can in some embodiments, make it dry with any Buffer fluid contacts described herein under capture agent presence or absence.

(but being not limited to) 2% tween (v/v) and 0.1%Triton (v/v) is comprised such as the example of damping fluid dissolving damping fluid; 2% tween (v/v) and 0.1%SDS (w/v); 2% tween (v/v) and 0.1%BSA (w/v); 2% tween (v/v) and 1%BSA (w/v); 0.1%SDS (w/v), 1%BSA (w/v) or its any combination.Dissolving damping fluid can also be such as 5% tween/PBS; 2% tween/PBS+0.1%SDS; 2% tween/PBS+1%BSA.Other examples dissolving damping fluid include, but is not limited to 5% tween-80 (v/v); 5%Triton X-100 (v/v); 5%NP40 (v/v); 2% tween-80 (v/v); 2%Triton X-100 (v/v); 2%NP40 (v/v); 1% tween-80 (v/v); 1%Triton X-100 (v/v); With 1%NP40 (v/v).Other components of sanitising agent (washing agent) and damping fluid can be prepared by any suitable buffer being applicable to protein and be included, but is not limited to water and phosphate buffered saline (PBS).Dissolving damping fluid can for the preparation of sample before sample contacts with device described herein.In some embodiments, dissolving damping fluid is not used.When hope detects surface protein or surface analysis thing in method, sample does not use dissolving damping fluid.Therefore, in some embodiments, sample maybe will cause cytolytic condition without undergoing dissolving.When using cell, cell can for the part of bridge joint mixture and alternative as being shown in the amplicon in Fig. 3.Cell through mark or without mark, can produce the capture agent of similar bridge joint mixture as long as exist.

Present subject matter also provides and detects the method for multiple analytes, comprises and makes sample and device as described herein and/or system contacts, wherein sample contacts pad and test membrane, wherein indicate the existence of multiple analytes with the positive reaction of test membrane.In some embodiments, pad comprises detecting signal unit or is incorporated into the capture agent of detecting signal unit.In some embodiments, test membrane comprises the second analyte specificity capture agent.This capture agent can be incorporated into the interaction unit be present on analyte.Sample can have the amplicon that such as difference (difference, differentially) marks.Such as, test membrane can comprise the first capture agent being incorporated into interaction unit existing on the first analyte.Pad can have the capture agent being incorporated into detecting signal unit.Be applied to device and contact pad and/or test membrane before, analyte can be hatched together with bridge-jointing unit and/or detecting signal unit.When the mixture of analyte, capture agent and detecting signal unit exists, instruction positive reaction.Otherwise, do not produce signal.Capture agent can be applied to test membrane, and make when it is incorporated into its specific binding partner, it will indicate positive reaction.System and device can be utilized to form mixture described herein.Such as, after the PCR reaction that the amplified production producing difference mark occurs, product and may be used for producing bridge joint mixture antibody together with hatch.Mixtures incubated is added in device subsequently.Sample flows through the pad containing capture agent and interacts with the test membrane containing another capture agent subsequently.In some embodiments, pad is removed or moves, and makes it possible to signal be detected.The movement of pad in vertical current device is described herein.If all analytes all exist, so produce bridge joint mixture and can single signal be detected.The specificity capture agent can used by any way on test membrane makes when detection reagent, and it can form line, circle, plus sige, dotted line, " X " or any other pattern.In some embodiments, the control line that indicating unit suitably works will cross over analyte specificity line and when multiple analytes exists and be detected, detectable label will form plus sige.Can by detecting detection reagent as described herein and determining to detect by the ordinary method that those skilled in the art is known.Similar approach may be used in such as ELISA system.

In some embodiments, sample contacts device, subsequently after sample contact device, is applied to device by damping fluid.Such as, the sample comprising analyte can contact with pad and make sample transfer to pad.After contacting with pad, another solution can be applied to device and flow vertically through device described herein to promote or to start.

In some embodiments, as described herein, capture agent is antibody.In some embodiments, the sample tested is solution, but also can be solution or damping fluid and the mixture of solid material that can be applied to device.Solution will dissolve one or more analytes subsequently and allows the capture agent of pad to contact with the suitable analyte be present in sample.In some embodiments, sample comprises cell lysates.In some embodiments, cell lysates obtains clarification to remove soluble material by centrifugal or other modes.

In some embodiments, method comprises and test sample is contacted with sample divider and sample divider is contacted with device.Test sample can for comprising the sample of the amplicon produced by multiple analytes.In some embodiments, method comprises makes sample divider and solution or Buffer fluid contacts, and wherein solution or damping fluid are applied to device.In some embodiments, before sample contacts with test membrane, sample contacts with pad.In some embodiments, sample contacts with test membrane with pad simultaneously.

In some embodiments, method comprises makes the pad of device described herein move, and wherein the mobile test membrane that exposes of device is for detection.In some embodiments, locking component moves pad.In some embodiments, pad is connected to locking component and/or sliding button component.In some embodiments, moving or removing of removable member can be moved or remove pad.In some embodiments, pad is connected to removable member and/or adhesion component.In some embodiments, when removable member moves or remove, adhesion component also moves about its starting position or removes from device.Any other analyte that analyte maybe can be able to use method and apparatus described herein to detect for the analyte discussed herein.In some embodiments, method comprises sample administration auto levelizer and allows sample to flow through device via vertical current.

In some embodiments, multiple analytes presence or absence detect or instruction be less than in 60 seconds occur.In some embodiments, the presence or absence of multiple analytes detects or instruction appearance in about 30 to about 60 seconds.In some embodiments, multiple analytes presence or absence detect or instruction be less than in 2 minutes occur.In some embodiments, the presence or absence of multiple analytes detects or instruction appearance in about 30 seconds.

In some embodiments, the device detecting multiple analytes with single signal is provided.In some embodiments, device comprises shell.Device can comprise the first casing component and second housing component to form shell.In some embodiments, the first and second casing components are individual member.First and second casing components can be fabricated to single-piece.In some embodiments, single-piece can be separated into two casing components to allow material to introduce in shell (such as device).In some embodiments, device comprises entrance.Entrance can in arbitrary casing component (such as, first or second housing component).Entrance can be oriented to above pad and make the sample in entrance introducing device contact pad before Contact test film.Through orientation, device makes no matter any pressure puts on device, sample all will flow through the layer of film (such as, detection of analytes membranous system) vertically downward.Therefore, in some embodiments, second housing component comprises import.In some embodiments, second housing component is at the top of the first casing component.Entrance can be any size as described herein or shape, as long as size and dimension is enough to make sample can survey membranous system by contact analysis quality testing in sample introduction device.

Device can comprise one or more afterburning component.Afterburning component can apply pressure to detection of analytes membranous system.Power is perpendicular to or is substantially perpendicular to film or the layer applying of detection of analytes membranous system.In some embodiments, device comprises at least 1,2,3,4 or 5 afterburning component.In some embodiments, device comprises at least 1,2,3,4 or 5 afterburning component.In some embodiments, device comprises multiple afterburning component.Afterburning component can contact with casing component.In some embodiments, the first surface of afterburning component and casing component (such as, first or second housing component) contact.In some embodiments, the second surface contact analysis quality testing of afterburning component surveys membranous system.As described herein, afterburning component may be used for compression detection of analytes membranous system to promote that sample flows through detection of analytes membranous system.Pressure flows through detection of analytes membranous system with can promoting samples vertical.Afterburning component can be oriented in device independently of one another.Afterburning component can also make each afterburning component apply pressure and the power putting on each detection of analytes membranous system is different or is identical or substantially the same in some embodiments to different detection of analytes membranous system through handling.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, device comprises one or more removable locking component.In some embodiments, removable locking component contacts afterburning component.In some embodiments, removable locking component contacts each the afterburning component be present in device.Such as, in the device comprising the first and second afterburning components, removable locking component and the first afterburning component and the second afterburning member contact.In some embodiments, removable locking component support afterburning component make afterburning component be improve position in.Can by comparing the position of determining with the position of component afterburning when afterburning component does not contact with removable locking component when afterburning component contacts with removable locking component to improve.Do not exist when contacting between afterburning component with removable locking component, afterburning component is in first location.When removable locking component and afterburning member contact, afterburning component is in the second position.In some embodiments, the second position of afterburning component is regarded as the position of raising.The position of improving may be used for the layer (such as, film) compressing detection of analytes membranous system.When removable locking component is not with afterburning member contact, in some embodiments, detection of analytes membranous system is not compressed.

Device can comprise one or more removable locking component.In some embodiments, device comprises multiple or 1,2,3,4 or 5 removable locking component.In some embodiments, device comprises at least 1,2,3,4 or 5 removable locking component.In some embodiments, the number of removable locking component that device comprises equals the number of the afterburning component be present in device.

Removable locking component can also comprise mobile member, such as (but being not limited to) handle.Mobile member may be used for such as rotating or move removable locking component and makes locking component contact afterburning component.In some embodiments, mobile member makes locking component depart from from afterburning component and makes afterburning component change position (such as, from the position mentioned to the position reduced).Mobile member may be used for removing applied pressure or apply described pressure on detection of analytes membranous system.Mobile member can also be used for the compression removing or apply detection of analytes membranous system.In some embodiments, mobile member makes locking component rotate around the central shaft of device.Such as, by sample administration in device and after sample flows through at least one detection of analytes membranous system, mobile member is moved, and it makes removable locking component rotate in the clockwise or counterclockwise direction.The rotation of removable locking component makes afterburning component be reduced to different positions.The rotation of removable locking component also allows to remove the pressure putting on detection of analytes membranous system.In some embodiments, pressure is fully removed, or in some embodiments, pressure is only partly removed.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, the mobile member of mobile removable locking component projects through first or second housing component.In some embodiments, can close to (accessible) mobile member through mobile member outlet.In some embodiments, the central shaft of mobile member around device when mobile rotates.In some embodiments, mobile member makes removable locking component laterally (such as, flatly) or vertically move.In some embodiments, removable locking component when mobile laterally (such as, flatly) or vertically move.

Mobile member and removable locking component can be constructed as single-piece or are two pieces.In some embodiments, wherein removable locking component and mobile member are independently two pieces and be configured to and interact with each otherly make the movement of one that another one is moved.Such as, one in two pieces can have " public component ", and male component is given prominence to from surface and inserted " female component " of another part to form interaction.

By mobile member, removable locking component is moved also to may be used for moving or remove the pad be present in detection of analytes membranous system.As discussed herein, pad can be removed to allow range estimation or analytical test film.As discussed herein, can fully remove pad from detection of analytes membranous system or remove pad with the amount being enough to permission range estimation or analytical test film.The analysis of test membrane can only based on visual control, or in some embodiments, and optical pickup can be used to carry out analytical test film to determine not existing or existing of antigen in sample.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, device comprises multiple or two or more detection of analytes membranous system.In some embodiments, device comprises at least 1,2,3,4 or 5 detection of analytes membranous system.In some embodiments, device comprises 1,2,3,4 or 5 detection of analytes membranous system.Detection of analytes membranous system can as herein with subject application in the whole text described by.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, device comprises one or more flexibility or inflexibility transom.In some embodiments, device comprises multiple flexibility or inflexibility transom.In some embodiments, device comprises at least 1,2,3,4 or 5 flexibility or inflexibility transom.In some embodiments, device comprises 1,2,3,4 or 5 flexibility or inflexibility transom.In some embodiments, flexible or inflexibility transom contacts removable locking component.In some embodiments, flexible or inflexibility transom contacts removable locking component and pad.Flexible or inflexibility transom may be used for removing or mobile pad away from the remainder of the layer (such as, film) of detection of analytes membranous system.In some embodiments, the number of the flexibility that comprises of device or inflexibility transom equals the number of the detection of analytes membranous system be present in device.In some embodiments, the number of flexible connecting member that device comprises equals the number of the afterburning component be present in device.Flexible or inflexibility transom also may be used for retraction pad to appear or to expose part or all of test membrane.

Such as, in some embodiments, device comprises three detection of analytes membranous system and three afterburning components.Have and may be used for detecting different analytes or different multiple analyte groups more than the device of a detection of analytes membranous system.In this kind of device, such as, device comprises first, second, and third transom.First transom can contact with removable locking component with the pad of the first detection of analytes membranous system.In addition, in some embodiments, the second transom can contact with removable locking component with the pad of the second detection of analytes membranous system.In some embodiments, the 3rd transom can contact with removable locking component with the pad of the 3rd detection of analytes membranous system.In some embodiments, first, second, and third transom contacts with identical removable locking component.In some embodiments, first, second, and third transom contacts with different removable locking components.Such as, in some embodiments, the first and second transoms to contact with identical removable locking component and the 3rd transom contacts with different removable locking components.Each transom contacts with one or more removable locking component independently.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, removable locking component comprises one or more removable locking component extension (extension).In some embodiments, removable locking component extension contacts afterburning component.In some embodiments, the number of removable locking component extension that comprises of device is identical with the number of the afterburning component be present in device.In some embodiments, removable locking component extension partly around or surround afterburning component.In some embodiments, removable locking component extension fully around or surround afterburning component.The shape of removable locking component or extension can remain on any shape in the position of raising for making afterburning component.In some embodiments, extension be partially or even wholly around or surround hook or the hook sample shape of afterburning component.Shape is not important, as long as shape is used as the upholder of power actuator (such as, afterburning component).

The number of removable locking component extension can be identical or different with the number of the afterburning component be present in device described herein.In some embodiments, device comprises multiple removable locking component extension.In some embodiments, device comprises at least 1,2,3,4 or 5 removable locking component extension.In some embodiments, device comprises 1,2,3,4 or 5 removable locking component extension.Such as, in some embodiments, device comprises first, second, and third afterburning component transom and first, second, and third removable locking component extension.In this limiting examples, such as, the removable locking component extension of the first afterburning member contact first, the removable locking component extension of the second afterburning member contact second, and the removable locking component extension of the 3rd afterburning member contact the 3rd.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, removable locking component comprises transom extension, and extension can be flexible or inflexibility.In some embodiments, transom extension contact transom.In some embodiments, transom extension comprises transom extension tubercle (node, nodule).Tubercle can for making transom firmly with any shape or the size that make transom maintain the contact of it and removable locking component securely.In some embodiments, one or more removable locking component extension extends from radially (such as, outside) of removable locking component.

The number of transom extension can be identical or different with the number of the detection of analytes membranous system be present in device described herein.In some embodiments, device comprises multiple flexibility or inflexibility transom extension.In some embodiments, device comprises at least 1,2,3,4 or 5 flexibility or inflexibility transom extension.In some embodiments, device comprises 1,2,3,4 or 5 flexibility or inflexibility transom extension.Such as, in some embodiments, device comprises first, second, and third transom and first, second, and third transom extension.In this limiting examples, such as, the first transom contacts the first transom extension, and the second transom contacts the second transom extension, and the 3rd transom contact the 3rd transom extension.

In some embodiments, device described herein comprises flexible and inflexibility transom and/or extension.In the present invention in the whole text, transom or the extension of flexibility or inflexibility can be mentioned.If an open flexible member of embodiment, so should be appreciated that, unless contrary instruction herein, otherwise also open wherein component is another embodiment of inflexibility.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, device comprises channel system.Channel system may be used for from the import traffic sample (such as, fluid) of device to the one or more detection of analytes membranous system be present in device.As used herein, " channel system " refers to whole system and does not consider have how many respective channel to be parts of system.Such as, channel system can comprise two or more from entrance by the passage of fluid transport to detection of analytes membranous system, such as (but being not limited to) kapillary.In some embodiments, channel system comprises one or more branch (such as, arm).One or more arm can by fluid transport to one or more detection of analytes membranous system.In some embodiments, channel system comprises 1,2,3,4 or 5 branch.In some embodiments, the number of the branch in the channel system that comprises of device equals the number of the detection of analytes membranous system be present in device.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, each branch of channel system comprises the kapillary of fluid transport to detection of analytes membranous system from entrance.In some embodiments, each branch comprises multiple kapillary.In some embodiments, each branch comprises at least 1,2,3,4 or 5 kapillary.In some embodiments, channel system does not comprise kapillary or pipe sample formation (formation), but by allowing the material of a pad part for sample being transported to analyte detection system from entrance to make.In some embodiments, channel system is porous material, and it may be used for, from entrance, sample is transported to detection of analytes membranous system.In some embodiments, channel system is made up of the material identical with pad.In some embodiments, channel system and pad are adjacent block of material.In some embodiments, channel system comprises Porex material.These porous materials allow entrance to be that fluid is communicated with detection of analytes membranous system.In some embodiments, porous material comprises polyethylene, polypropylene, tetrafluoroethylene (PTFE), PVDF, ethyl vinyl acetate (ethyl vinyl acetate), nylon 6 (Nylon 6), thermoplastic polyurethanes (TPU), SCP etc.In some embodiments, pad and channel system are same material or differing materials.In some embodiments, channel system does not comprise porous material and/or capillary system.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, channel system contact entrance.In some embodiments, channel system contact analysis quality testing surveys the top of membranous system.In some embodiments, the channel system contact top of pad or the film at pad top.In some embodiments, the edge of the channel system contact edge of pad or the film at pad top.With sample how contact analysis quality testing survey membranous system and have nothing to do, in some embodiments, flow through to samples vertical detection of analytes membranous system.Therefore, although sample flatly (such as, laterally) can flow to detection of analytes membranous system from entrance, not analytic sample, until it flows vertically through detection of analytes membranous system.This and wherein sample laterally (such as, flatly) to flow through the transverse flow system of multiple film or test layer obviously different.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, sample is divided into moiety by channel system, and wherein each moiety contacts independently detection of analytes membranous system.In some embodiments, sample is divided into one or more unequal part by channel system.One or more unequal part is transported to independently detection of analytes membranous system subsequently.

Such as, in the device comprising the first and second detection of analytes membranous system, device comprises the channel system comprising the first and second branches.In some embodiments, the first branch contacts the first detection of analytes membranous system and the second branch contacts the second detection of analytes membranous system.When by sample administration auto levelizer (such as, via import), sample is transported to the first and second detection of analytes membranous system with moiety through the first and second branches of channel system.In some embodiments, sample is transported to the first and second detection of analytes membranous system with unequal part through the first and second branches of channel system.Sample can be divided into unequal part, such as, based on the number of the kapillary be present in each branch.Such as, the first branch can comprise the kapillary more than the second branch.More the kapillary of high number will allow more Multi-example through the first branch but not second branch transport, send unequal part thus to the first and second detection of analytes membranous system.

Therefore, the branch of channel system can have the kapillary of similar number or the kapillary of different number.The number of the kapillary in each branch of channel system is independent of each branch.That is, each branch of channel system can have the kapillary of similar number or different number from another branch.Therefore, in some embodiments, the channel system of device can be described as capillary channel systems.In some embodiments, channel system is encapsulated in and separates about the first and second casing components that device itself is described herein and in different channel envelope.In some embodiments, channel envelope is transparent, translucent, opaque or part translucent.

As discussed herein, with human eye naked eyes or can pass through machine, such as optical pickup carrys out analytical test film with the presence or absence by single signal determination multiple analytes.In some embodiments, analysis is undertaken by port (portal).In some embodiments, device comprises one or more port, and its size is enough to the test membrane allowing the one or more detection of analytes membranous system of range estimation.In some embodiments, single-port is used to estimate each test membrane be present in device.In some embodiments, device does not comprise port.Device does not comprise in the embodiment of port wherein, and test membrane still can be estimated by using the transparent or semitransparent shell being used for device.In some embodiments, first and/or second housing be transparent or semitransparent.When first and/or second housing are transparent or semitransparent, this can allow detection of analytes membranous system and its test membrane to appear when moving or removing pad.In some embodiments, device comprises multiple port.In some embodiments, device comprises at least 1,2,3,4 or 5 port.In some embodiments, device comprises 1,2,3,4 or 5 port.In some embodiments, device comprises 1 port, and port is continuous print and exposes each detection of analytes membranous system of being present in device to carry out visual control.

As discussed herein, can allow afterburning component move between at least two positions (such as, raising or reduce; (joint, the engaged) that engage or (unassembled, the disengaging, disengaged) of not engaging).In some embodiments, afterburning component reduces and surrounded by power actuator exit.Therefore, in some embodiments, device comprises one or more power actuator exit, and when afterburning component reduces, outlet can accept afterburning component.In some embodiments, device comprises multiple power actuator exit.In some embodiments, power actuator exit is groove.In some embodiments, power actuator exit is circle or substantially circular.The power actuator exit actuator (such as, afterburning component) that may be used for exerting all one's strength is suspended on specific location.Power actuator exit can also be used for exerting all one's strength actuator maintenance in the second position.In some embodiments, the circumference of power actuator exit is greater than the circumference of the part entering outlet in afterburning component.In some embodiments, the circumference of power actuator exit is greater than the largest circumference of afterburning component.In some embodiments, the circumference of power actuator exit is not more than the largest circumference of afterburning component, and wherein afterburning component contains the region with at least two different circumference.Such as, describe herein and will have the afterburning component of two different circumference.Afterburning component can comprise the supporting structure of the cap with a circumference and the support cap with different circumference.Supporting structure can have the circumference less than cap in some embodiments.In some embodiments, power actuator exit can have and is greater than supporting structure circumference but the circumference being less than cap structure circumference.In some embodiments, the number of power actuator exit is identical or different with the number being present in the afterburning component in device.

Power actuator exit can also be the continuous depression (depression) in casing component, when the afterburning component of each in device reduces and when not recompressing detection of analytes membranous system, outlet can accept afterburning component.Outlet may be used for temporarily holding afterburning component, or it can be permanent, makes afterburning component cannot improve to compress or compress detection of analytes membranous system further again.

As herein with subject application in the whole text discuss, pad, permeable membrane, test membrane and absorption component can by or by afterburning component as described herein and compress under including, but is not limited to some power of the power of about 1lbf to about 100lbf.In some embodiments, wherein there is multiple detection of analytes membranous system, the pressure putting on each film detection system can be different, or it can be identical.Such as, in the device with first, second, and third detection of analytes membranous system, first detection of analytes membranous system can be compressed under the power of 5lbf, second detection of analytes membranous system can be compressed under the power of 10lbf, and the 3rd detection of analytes membranous system can be compressed under the power of 25lbf.In another example, in some embodiments, the first and second detection of analytes membranous system are compressed at the same pressure and the 3rd detection of analytes membranous system is compressed under the pressure being different from the first and second detection of analytes membranous system.The difference of pressure may be used for using different flow rates, and different flow rates goes for different analytes.Pressure is relevant to flow rate.Pressure can be handled by the number of plies by compression in the position of afterburning component and detection of analytes membranous system.Known and the method for routine can be used to determine and measure used certain force by those skilled in the art.

As described herein, in some embodiments, the invention provides the system comprising any device described herein, buffer container and/or sample divider.In some embodiments, the invention provides one or more the test kit comprised in any device described herein and positive control, negative control thing, specification sheets, buffer container and/or sample divider or its any combination.

Method described herein can use together with having the device of such as multiple (two or more) detection of analytes membranous system.Method can also use together with having the device of 2,3,4 or 5 detection of analytes membranous system.Existence more than two detection of analytes membranous system (such as, 3,4,5,6,7,8,9 or 10) time, revise method comprised herein and describe with consistent with the number of detection of analytes membranous system.These change be according to description comprised herein and those skilled in the art known any routine change carry out.Excessive experiment will do not needed more than the change of 2 analyte film detection systems herein about containing based on the knowledge of comprised description and those skilled in the art.In embodiments more as described herein, device comprises two or more detection of analytes membranous system.In some embodiments, method comprises and makes sample (such as, comprising the sample of multiple analytes) contact with device and through channel system, a part of sample is transported to the pad of two or more detection of analytes membranous system.In some embodiments, method comprises the positive or negative reaction of detect analytes, and wherein positive reaction instruction exists multiple analytes.In some embodiments, two or more detection of analytes membranous system are compressed by afterburning component.In some embodiments, sample vertically flows to test membrane from pad.In some embodiments, method comprises further by afterburning component compression detection of analytes membranous system.In some embodiments, method is included in a part of sample and has contacted and after flowing through the pad of two or more detection systems, pad moved, and exposes test membrane thus for analysis.In some embodiments, test membrane is exposed to for detecting in port openings (portal opening).In some embodiments, the pad of two or more detection systems moves by making removable locking component.In some embodiments, removable locking component is moved comprise removable locking component is rotated around the central shaft of device.In some embodiments, removable locking component laterally or is vertically moved.In some embodiments, the pad of removable locking component simultaneously or sequentially two or more detection systems mobile.In some embodiments, method comprises the compression removing two or more analyte detection system further.Can such as remove by making removable locking component move or reduce pressure.In some embodiments, removable locking component be mobile member by making to be connected to removable locking component rotate or mobile come (such as, the rotating) of movement.

In some embodiments, one or more detection of analytes membranous system was compressed before sample contacts channel system.In some embodiments, one or more detection of analytes membranous system was compressed before the pad of the one or more detection of analytes membranous system of sample contacts.In some embodiments, each detection of analytes membranous system is compressed simultaneously.In some embodiments, each detection of analytes membranous system is compressed independently.In some embodiments, each detection of analytes membranous system be present in device was compressed before sample contacts pad.

In some embodiments, method comprises to be made two or more detection of analytes membranous system are removed by afterburning component applied pressure, and wherein said afterburning component vertically or flatly moves to remove described pressure.In some embodiments, method comprises and makes afterburning component move to the second position with pressure relief from first location.In some embodiments, afterburning component moves in power actuator exit or with power actuator exit and contacts, now the mobile power removed or reduce that pressure or releasing or minimizing put on detection of analytes membranous system of afterburning component.In some embodiments, afterburning component partially or even wholly exits (drop out) device.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, the invention provides the device for detect analytes, it comprises pressure actuator, Pressure-relief Device, detection of analytes membranous system, detection of analytes membranous system susceptor and outlet.In some embodiments, detection of analytes membranous system susceptor (receptacle) is for being enough to the size and dimension accepting detection of analytes membranous system.In some embodiments, susceptor is groove.In some embodiments, susceptor is can (but nonessential) box of removing from device.

In some embodiments, detection of analytes membranous system as described herein can be enclosed in or be contained in cylinder or shell.Shell can comprise first and/or second housing component.In some embodiments, wherein detection of analytes membranous system is contained in shell or cylinder, and susceptor has the size and dimension being enough to accept shell or cylinder.In some embodiments, shell or cylinder comprise entrance.Entrance may be used for using sample to detection of analytes membranous system.In some embodiments, cylinder or shell comprise the second outlet allowing sample to flow through and flow out shell and cylinder.Detection of analytes membranous system can be any detection of analytes membranous system as described herein.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, device comprises pressure actuator.Such as, pressure actuator can be afterburning component described herein.In some embodiments, pressure actuator is pressure lock or vacuum valve, its to detection of analytes membranous system apply air pressure or by analysis quality testing survey membranous system vacuumize.In some embodiments, pressure actuator can be adjusted by Pressure-relief Device or pressure regulator.Pressure-relief Device or pressure regulator can be such as vacuum release.Releasing device or regulator may be used for adjustment to detection of analytes membranous system applied pressure or vacuum.Pressure or vacuum can by being present in outlet in device or pipe puts on detection of analytes membranous system.Outlet can for being present in the identical outlet in described cylinder or shell herein, or it can be different outlets or pipe.Outlet or pipe can be used to make pressure or vacuum will apply with specificity but not make it spread in whole device.

In some embodiments, the shell (such as, cylinder) that coated analyte film detects comprises upper case and lower case.In some embodiments, shell comprises multiple film or pad clamper.In some embodiments, shell comprises one or more film or pad clamper.In some embodiments, shell comprises 1,2,3,4 or 5 film or pad clamper.In some embodiments, shell comprises at least 1,2,3,4 or 5 film or pad clamper.In some embodiments, shell comprises entrance.In some embodiments, shell comprises outlet.In some embodiments, vacuum actuator directly or indirectly contacts housing outlet.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, device and any device described herein all comprise the injector for spraying shell.Injector may be used for promoting removing of the shell containing detection of analytes membranous system.In some embodiments, device comprises shell separator.Shell separator may be used for the separation promoting shell.In some embodiments, injector also can be used as shell separator.

Except method described herein, in some embodiments, detect the method for multiple analytes also to comprise and use sample containing multiple analytes to the device comprising pressure actuator, pressure regulator, detection of analytes membranous system, detection of analytes membranous system susceptor and outlet or any other device described herein or detection of analytes membranous system.In some embodiments, sample contacts with detection of analytes membranous system, wherein flows through detection of analytes membranous system to samples vertical.In some embodiments, method comprises the presence or absence of detect analytes.This can carry out according to the bridge joint mixture by using interaction unit described herein, capture agent and detecting signal unit to be formed.

Detect in some embodiments of multiple analytes at using appts, detect comprise remove or amount that the mobile pad be present in detection of analytes membranous system is enough to make result visible, wherein positive findings indicates the existence of multiple analytes.In some embodiments, detect comprise remove detection of analytes membranous system from device and remove further or amount that mobile pad is enough to make single signal to the detection of analyte or multiple analytes.In some embodiments, detection of analytes membranous system is contained in shell or cylinder, and therefore, and in some embodiments, shell or cylinder are mobile or remove from device before removing pad.In some embodiments, to remove or before mobile pad, shell is separated into first (such as, top) and second (such as, bottom) shell as described herein.In some embodiments, shell be separated into the first and second shells can remove or mobile pad to make test membrane as described herein visible.In some embodiments, shell manually and/or is mechanically separated.In some embodiments, shell (such as, cylinder) is ejected by from device.In some embodiments, shell is ejected from device by by injector.In some embodiments, shell is separated by by separator.In some embodiments, injector is also used as separator.

In some embodiments, method is included in and detection of analytes membranous system applies pressure or vacuumized by detection of analytes membranous system.In some embodiments, method comprises release or reduces pressure or vacuum.In some embodiments, by using pressure regulator discharge or reduce pressure or vacuum.In some embodiments of method described herein, the sample contacted with detection of analytes membranous system flows through analyte membranous system with the flow rate adjusted by pressure actuator.In some embodiments, whole sample flows through detection of analytes membranous system with constant rate of speed.In some embodiments, sample flows through detection of analytes membranous system with variable bit rate.In some embodiments, variable bit rate is included in the flow rate of a part of sample at least for some time is 0.Such as, institute's applied pressure or the vacuum of taking out can be adjusted make sample stop running through detection of analytes membranous system to continue for some time.This can be called " stopping (dwell) ".As in document of the present invention described by all the other places, can realize stopping by between other layers of not saturating or micro-permeable membrane (slightly impermeable membrane) being placed on pad and detection of analytes membranous system.But, also can put on the pressure of detection of analytes membranous system to adjust stop by adjustment (such as, changing).Also the vacuum can taken out by detection of analytes membranous system by adjustment (such as, changing) adjusts stop.Can Use Adjustment by any method of the flow rate of detection of analytes membranous system, include, but is not limited to the flow rate by pad and/or test membrane.

Device herein also can be automatization or be combined with the spectrometer of optical pickup or other types.The susceptibility that the advantage that the spectrometer of system described herein and device and optical pickup or other types is combined is to increase device and analysis makes for being less to the analyte necessary analyte be present in sample that is positive by sample identification.This larger susceptibility can subsequently for such as determining whether that food contains pathogenic agent, whether people suffers from certain disease or the patient's condition, or whether product has and use other apparatus and method using the analyte that cannot detect in the same time amount spent by the method and apparatus that describes at present in other cases.

Therefore, may be used for detecting in some embodiments of the device of multiple analytes with single signal, the device that the invention provides for detecting multiple analytes comprises sample inlet, detection of analytes socket joint receiver, detection of analytes socket joint receiver entrance, optional pad remover, pressure actuator, spectrometer (such as, optical pickup), display unit (display unit), signal processing unit.Pressure actuator can be afterburning component, and its position is applied to the pressure of the detection of analytes membranous system be combined with device with adjustment through amendment.Pressure actuator can also vacuumize through the detection of analytes membranous system be combined with device and adjust pressure.Spectrometer can be can any spectrometer of existence of detection signal.Signal can be optical signalling.Signal can for be selected from the signal launched in the spectrum of such as infrared spectra, near infrared spectrum, visible spectrum, x-ray spectrum, ultraviolet spectrogram, gamma-rays, electromagnetic spectrum etc.

Spectrometer can be connected to signal processing unit (such as, computer).Signal processing unit can obtain to its transmit signal and analytical signal to determine the presence or absence of sample.The example of signal processing unit is (but being not limited to) computer.Signal processing unit can sequencing to analyze the signal that transmitted by spectrometer.Sequencing can carry out formula with analytical signal, thus determines the presence or absence of analyte in sample.Formula can based on pre-installation in the storer of signal processing unit or the criterion that inputted by the user of device.The type of the information that can input can be the cut-off (cut-off), treatment time etc. of positive or negative signal.Signal processing unit can also be used for adjusting to detection of analytes membranous system applied pressure or the vacuum of being taken out by detection of analytes membranous system.

Signal processing unit may be used for or through sequencing to adjust the flow rate that sample flows through detection of analytes membranous system.Flow rate can be adjusted to detection of analytes membranous system applied pressure or by the vacuum that detection of analytes membranous system is taken out by adjustment.As above about described by method described herein, the sample contacted with detection of analytes membranous system flows through analyte membranous system with the flow rate adjusted by pressure actuator.Pressure regulator can be adjusted by such as signal processing unit conversely.In some embodiments, whole sample flows through detection of analytes membranous system with constant rate of speed, and speed is adjusted by signal processing unit.In some embodiments, sample flows through detection of analytes membranous system with variable bit rate, and speed is adjusted by signal processing unit.In some embodiments, the flow rate that variable bit rate is included in a part of sample at least for some time is 0, and flow rate can be adjusted by signal processing unit.Such as, institute's applied pressure can be adjusted by signal processing unit or the vacuum of taking out makes sample stop running through detection of analytes membranous system, continue for some time.As discussed herein, this can be called " stop ".Such as, can put on the pressure of detection of analytes membranous system to adjust stop by adjustment (such as, changing), adjustment can be realized by signal processing unit or control.Also the vacuum can taken out by detection of analytes membranous system by adjustment (such as, changing) adjusts stop, and adjustment can be realized by signal processing unit or control.The flow rate of detection of analytes membranous system can be flow through by Use Adjustment, include, but is not limited to any method of the flow rate flowing through pad and/or test membrane, and method can be adjusted by signal processing unit or realize.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, device described herein and in the whole text comprises detection of analytes socket joint receiver align member (positioning member).Detect socket joint receiver align member to may be used for such as detection of analytes membranous system being placed in appropriate location to accept sample and/or to be used for sample to be analyzed.In some embodiments, system is through locating for spectrometer analysis.In some embodiments, and/or can control to detect socket joint receiver align member by signal processing unit motorize (motorized).In some embodiments, detect socket joint receiver align member without motorize, but the lever of the position of amendment susceptor or screw rod can be allowed to control by hand controller such as (but being not limited to).In some embodiments, signal processing unit moves by making analyte film detect susceptor mobile member the movement that control analysis thing film detects susceptor.In some embodiments, detection of analytes socket joint receiver align member contacts with detection of analytes socket joint receiver.

May be used for detecting in some embodiments of the device of multiple analytes with single signal, device described herein can comprise waste receptacle.Waste receptacle can in the inside of device or in outside, but still contact device.Waste receptacle can receive the detection of analytes membranous system used.In some embodiments, as described herein, detection of analytes membranous system is contained in shell (such as, cylinder).Shell can be injected in waste receptacle subsequently.Injection can be manual or automatization.In some embodiments, injection is controlled by signal processing unit.In some embodiments, signal processing unit controls injector, and detection of analytes membranous system is ejected into waste receptacle from detection of analytes membranous system susceptor by injector.All devices as described in this article, in some embodiments, device comprises detection of analytes membranous system, and it can be included in or can not include in shell (such as, cylinder).

May be used for detecting in some embodiments of the device of multiple analytes with single signal, pressure actuator contact analysis quality testing surveys membranous system.In some embodiments, pressure actuator is connected to device at the some place of authorized pressure actuator movement.In some embodiments, pressure actuator connects in the pivot point of authorized pressure actuator in single point of contact place pivotable.

In some embodiments, device described herein comprises indicating meter.In some embodiments, indicating meter is electronic console.In some embodiments, signal processing unit accepts the input from spectrometer and shows information on the display unit.Display unit can show various information, includes, but is not limited to the existence of one or more analyte, states etc. and/or does not exist.

In some embodiments, the present invention includes to use and comprise the device of signal processing unit or device described herein to detect multiple analytes.In some embodiments, method comprises makes device and sample contacts, the detection of analytes membranous system in sample contacts device.Sample flows through detection of analytes membranous system subsequently.In some embodiments, method comprises the presence or absence of detect analytes.In some embodiments, detect and comprise the optical signalling of optical pickup detection from analyte membranous system, optical signalling is communicated to signal processing unit by optical pickup, and signal processing unit analyzes optical signalling to determine the presence or absence of analyte; Result is shown on the display unit with signal processing unit.Shown result can be vision and/or the sense of hearing.The signal analyzed can for the signal in the spectrum being selected from infrared spectra, near infrared spectrum, visible spectrum, x-ray spectrum, ultraviolet spectrogram, gamma-rays or electromagnetic spectrum.In some embodiments, signal is near infrared spectrum.In some embodiments, method comprises and being ejected in waste receptacle by detection of analytes membranous system.In some embodiments, signal processing unit is computer.

With reference to accompanying drawing, in some embodiments, the embodiment of Fig. 1-Figure 36 drawing apparatus, the assembly of representative device and to may be used in method and/or with other devices described herein and/or system is combined or the various views of specific device that are not combined.

These devices described herein are nonrestrictive and any other device (comprising other vertical current devices) all can be used for using the bridge joint mixture utilizing multiple mark and capture agent to produce to detect multiple analytes according to method described herein.

Fig. 8 describes to may be used for detecting the device of multiple analytes with single signal, comprises the first casing component (10), buffer container (15), second housing component (20), groove (25) for sliding button, sliding button (30), import (35), hoop (40) and test membrane (45).Fig. 8 describes the test membrane (45) comprising two kinds of capture agents.First (10) and second (20) casing component also can be called bottom and upper case component.In FIG, sample will be used by import (35) and it can be made vertically to flow to test membrane (45).In fig. 8, groove (25) allows sliding button to move, and sliding button is moving locking member when being connected to locking component, and can move pad in some embodiments and change the position of afterburning component.

Fig. 9 describes the device that may be used for detecting multiple analytes with single signal, comprise the first casing component (10), second housing component (20), groove (25) for sliding button, sliding button (30), import (35), hoop (40), test membrane (45), pad (50), multiple absorption component (such as, padding) (55), transom (60), locking component (65) and afterburning component (70).Fig. 9 describes pad (50), test membrane (45) and absorption component (55) substantially arranged parallel to each other.Afterburning component (70) will apply the pressure being substantially perpendicular to pad when contacting with absorption component.As shown in Figure 9, the sample contacted with device by import (35) will be passed vertically through pad (50) and flow to test membrane (45).Although clearly do not show in Fig. 9, but in some embodiments, permeable membrane is also arranged essentially parallel to pad (50) and test membrane (45), the wherein surface of first surface contact pad (50) of permeable membrane, the surface of the second surface Contact test film (45) of permeable membrane.

Figure 10 describes pad (50), permeable membrane (75), test membrane (45) and multiple absorption component (55), and it may be used for detecting multiple analytes with single signal.Figure 10 describes the assembly that may be used for detecting multiple analytes with single signal, and it is substantially parallel to each other.Figure 10 describes the permeable membrane (75) comprising opening.This opening can be used to allow the visual of the result of test membrane and detect.

Figure 11 describes the device that may be used for detecting multiple analytes with single signal, comprise the first casing component (10), buffer container (15), second housing component (20), sliding button (30), test membrane (45), pad (50), permeable membrane (75), multiple absorption component (such as, padding) (55), transom (60), locking component (65) and afterburning component (70).Figure 11 also describes the afterburning component (70) comprising axle (72) and head (71), and wherein head (71) is wider than axle (72).

Figure 12 describes the partial view that may be used for the device detecting multiple analytes with single signal, comprises the first casing component (10), locking component (65), sliding button (30) and afterburning component (70).The locking component (65) that Figure 12 describes to contact with afterburning component (70) makes afterburning component (70) in the method improved.Figure 12 also describes the movement away from afterburning component (70) of locking component (65) and sliding button (30), allows afterburning component to change position.In some embodiments, the change of position is that afterburning component reduces.

Figure 13 describes the side sectional view that may be used for the device detecting multiple analytes with single signal, comprises the first casing component (10), second housing component (20), sliding button (30), locking component (65), hoop (40), O type ring (41), afterburning component (70) and the upholder (73) for afterburning component.Upholder for axle can be a part for such as the first casing component (10) and only differentially draw shade for example object.Figure 13 describes the button (30) contacted with locking component (65), and its way of contact makes the movement of button (30) by moving locking member (65).The movement of locking component (65) will take away upholder from afterburning component (70), and this will allow afterburning component (70) to change position.Figure 13 also describes axle (72) and the head (71) of afterburning component.Head (71) produces lip, and wherein locking component (65) can support afterburning component (70) in afterburning component (70) slid underneath.

Figure 14 describes the partial view that may be used for the device detecting multiple analytes with single signal, comprises the first casing component (10), second housing component (20), import (35), test membrane (45), pad (50), multiple absorption component (55), transom (60), locking component (65) and afterburning component (70).Fig. 8 describes the transom (60) being connected to pad (50) and locking component (65).Figure 14 also describes against the compressed pad of the circumference of second housing component (20) and import (35).Figure 14 describes the head (71) executing stressed afterburning component by contacting multiple absorption component (55).In fig. 14, sample is applied to device by import (35), makes sample contacts pad (50) and due to pressure, and sample is by vertical current Contact test film (45).

Figure 15 A describes the partial view that may be used for the device detecting multiple analytes with single signal, comprises the first casing component (10), second housing component (20), import (35), test membrane (45), pad (50), multiple absorption component (55), transom (60), locking component (65) and afterburning component (70).Fig. 8 describes the movement of locking component (65), and locking component (65) is connected to transom (60).The movement being connected to the transom (60) of pad (50) makes pad move.Figure 15 A describes to change the afterburning component (70) of test of position and the pressure of test membrane (45) and/or alleviating or eliminating of compression.Figure 15 C and Figure 15 D also describes the movement of pad (50) away from import (35), appears test membrane (45) for visual and/or detection.

Figure 16 describes the transom (60) being connected to pad (50).Figure 16 describes the recess (51) of the position connected as transom (60) in pad (50).Transom also can by other means as being connected with other types of attachment by tackiness agent, bail.

Figure 17 describes the partial view that may be used for the device detecting multiple analytes with single signal, comprises second housing component (20), multiple pad or film (80) (wherein multiple pad comprises test membrane, permeable membrane and one or more absorption component) and can keep the retaining member (85) of multiple pad or film (80).Figure 10 describes multiple pad when mobile pad and is retained in the structure in appropriate location.Any mode or other structures all can be used for multiple pad is remained in appropriate location.

Figure 18 describes the representative device that may be used for detecting multiple analytes with single signal, comprises the first casing component (1002) and second housing component (1004) that comprise housing entry (1006) further.In some embodiments, the first and second casing components can be constructed as single unit.Housing entry allows sample to introduce in the assembly of enclosure.Housing entry can have the size of the appropriate volume being enough to dispose the solution added in device.In some embodiments, the size of the opening produced by housing entry is enough to dispose about 0.1 to about 3ml, about 0.1 to about 2.5ml, about 0.5 to about 2.0ml, about 0.1 to about 1.0ml, about 0.5 to about 1.5ml, about 0.5 to about 1.0ml and about 1.0 to about 2.0ml.In some embodiments, the size of device makes any size (such as, width, the degree of depth or height) all be less than or equal to about 5.08cm (2.000 inches).In some embodiments, the height of device is less than about 0.635cm (0.250 inch), is less than about 0.254cm (0.100 inch), is less than about 0.191cm (0.075 inch), is less than about 0.165cm (0.065 inch), is less than about 0.152cm (0.06 inch) or is less than about 0.140cm (0.055 inch).In some embodiments, the height of device is about 0.127cm (0.050 inch).In some embodiments, the width of device or the degree of depth are less than or equal to about 5.08cm (2.000 inches), about 4.83cm (1.900 inches), about 4.699cm (1.850 inches), about 4.572cm (1.800 inches), about 4.445cm (1.750 inches), about 4.191cm (1.650 inches), about 4.064cm (1.600 inches) or about 3.81cm (1.500 inches).In some embodiments, the height of device is about 0.127cm (0.050 inch), and the degree of depth is about 4.445cm (1.750 inches), and width is about 3.81cm (1.500 inches).

In some embodiments, may be used for detecting the device of multiple analytes with single signal and comprise multiple assembly, assembly comprise following one or more: removable member, pad, adhesion component, test membrane, absorption component, afterburning component, bracing member or its any combination.

In some embodiments, the device that may be used for detecting with single signal multiple analytes comprises afterburning component, removable member, pad, test membrane, adhesion component and/or absorption component.In some embodiments, device comprises detection of analytes membranous system.In some embodiments, detection of analytes membranous system comprises pad, test membrane and absorption component.In some embodiments, detection of analytes membranous system comprises other permeable membrane, but device also can not contain permeable membrane.In some embodiments, detection of analytes membranous system comprises in the following order: pad, adhesion component, test membrane and absorption component.

Figure 19 describes the exploded view that may be used for the inside detecting the representative device of multiple analytes with single signal, device comprises removable member (1005), pad (1050), adhesion component (1010), test membrane (1030), absorption component (1040) and bracing member (1020), and wherein bracing member comprises optional bracing member entrance (1025) further.Removable member and adhesion component also can comprise optional removable member entrance (1008) and adhesion means inlet (1012) respectively.Assembly can in the device of such as Figure 18.

Figure 20 describes the representative modul that may be used for another representative device detecting multiple analytes with single signal, comprises adhesion component (1010), bracing member (1020), test membrane (1030) and absorption component (1040).As shown in Figure 20, sample can flow through adhesion component (1010) and Contact test film (1030).

Figure 21 describes adhesion component (1010), bracing member (1020), test membrane (1030) and absorption component (1040).It is substantially parallel to each other that Figure 21 describes assembly.Figure 21 describes the bracing member (1020) comprising bracing member entrance (1025) further.This entrance may be used for flowing through device with allowing samples vertical.

Figure 22 partly describes pad (1050), test membrane (1030) and absorption component (1040).Figure 22 also describes the pad contacting and/or be connected to removable member (1005).Figure 22 also describes just to remove or the removable member of movement away from may be used for detecting the device of multiple analytes with single signal, and it also makes pad remove away from device or movement.The mobile test membrane that allows of pad is visualized, and this promotes, with single signal analysis and resolution analyte, to comprise multiple analytes.

Figure 23 describes the example of afterburning component (such as, fixture).Representative afterburning component can be various shape, size and configuration, but each component all applies pressure being positioned on the assembly in afterburning component or on afterburning component.Each afterburning component can also comprise opening (+), and analyte sample is used wherein.Figure 23 describes the limiting examples with the afterburning component of the first component (110) and second component (100).

Figure 24 A, Figure 24 B, Figure 24 C and Figure 24 D partly describe to comprise the afterburning component of the first component (110), b) second component (100), entrance (115) and detection of analytes membranous system (120).Figure 24 A and Figure 24 B also partly describes pad (1050).Pad is invisible in Figure 24 C and Figure 24 D.Figure 24 C and Figure 24 D also partly describes the test membrane (1030) of the part as detection of analytes membranous system.Figure 24 D also partly describe by colour band visual with the test membrane (1030) of control responses.

Figure 25 partly describes to comprise the container of trim plate of removable or removable trim plate (200), entrance (210), pad (1050) and pad (1050).The trim plate (255) of pad may be used for removing pad (1050) to expose test membrane from device.Such as, user can extract the trim plate (255) of pad out to remove pad (1050) from container.What do not estimate is the detection of analytes membranous system compressed between the first component (110) as described herein and second component (100).

Figure 26 partly describes the first external member (310), the second external member (320), removable or removable trim plate (330) and pad (1050).Removable or removable trim plate (330) comprises entrance, and entrance exposes pad (1050), makes sample to be applied to pad.Figure 26 does not show the first internals (110) and second internals (100) of compression detection of analytes membranous system (120).Removable or removable trim plate (330) is in movement or can move or remove pad (1050) when removing, and this allows range estimation and analytical test film.

Removable member entrance in removable member allows sample to introduce on pad.Entrance can have the size of the appropriate volume being enough to dispose the solution added in device.In some embodiments, the size of entrance is even as big as disposing about 0.1 to about 3ml, about 0.1 to about 2.5ml, about 0.5 to about 2.0ml, about 0.1 to about 1.0ml, about 0.5 to about 1.5ml, about 0.5 to about 1.0ml and about 1.0 to about 2.0ml.Removable member also can make that a part for removable member is permeable (that is, the region limited by removable member entrance) to solution and another region is impermeable through building.Thoroughly can be used as entrance in region, because permission solution is contacted pad by removable member by it.Removable member entrance can have any one in various shape and size.In some embodiments, the first casing component is used as removable member.In other embodiments, the first casing component and removable member are stand-alone assembly.First casing component and removable member are in the embodiment of stand-alone assembly wherein, and the overlap at least partially of housing entry and removable member entrance makes solution can enter two entrances.

In some embodiments, the first surface of removable member contact pad.Removable member also can be connected to pad.Removable member can by any mode be connected to pad make when removable member from device remove or its position change time, pad is also removed or the position of pad also changes.Removable member can be connected to pad by such as (but being not limited to) tackiness agent.Tackiness agent includes, but is not limited to other materials that glue, adhesive tape maybe will allow removable member and pad to be connected to each other.

In some embodiments, removable member directly contacts pad or contacts pad indirectly by another layer.Sample directly can be applied to pad by the opening in removable member in some embodiments.

The vertical view that may be used for the device detecting multiple analytes with single signal is described on Figure 27 part A ground, comprises multiple port (2036), entrance (2035) and casing component (2010).Figure 27 A also partly describes the part by port (2301) visible channel system (2300).The magnification region of Figure 27 part B ground drawing apparatus is port (2036) particularly.In port, also show multiple kapillary (2301).

Figure 28 describes the fish-eye view that may be used for the device detecting multiple analytes with single signal, and device comprises multiple power actuator exit (2200), casing component (2020) and mobile member (2100).

Figure 29 partly describes the first casing component (2010), second housing component (2020), multiple port (2036), entrance (2035), channel system (2300), multiple kapillary (2301), pad (2050), multiple test membrane (2045) and removable locking component (2065).The channel system described in Figure 29 is depicted as and is made up of 3 branches, and branches equals the number of the detection of analytes membranous system be present in device.

Figure 30 partly describes second housing component (2020), channel system (2300), multiple kapillary (2301), pad (2050), test membrane (2045) and absorption component (2055) and removable locking component (2065), flexible connecting member (2060), detection of analytes membranous system (2400).

Multiple power actuator exit (2200), channel system (2300), multiple kapillary (2301), multiple afterburning component (2070), removable locking component (2065), multiple removable locking component extension (2068), pad (2050), multiple flexibility or inflexibility transom extension (2066) and tubercle (2067), test membrane (2045) and absorption component (2055) are described in Figure 31 part A ground.

The similar portions of the device of Figure 31 part B ground shown in depiction 24A, but, removable locking component (2065) rotates around central shaft, and removable locking component extension (2068) no longer supports afterburning component (2070), and afterburning component is returned or drop in power actuator exit (2200).

Figure 32 partly describes to may be used for the exploded view of the device detecting multiple analytes with single signal, and device comprises channel system (2300), pad (2050), test membrane (2045), multiple afterburning component (2070), can rotate the movable member (2100) of described removable locking component (2065).Figure 32 also partly describes a part (2047) for removable locking component extension (2068), multiple flexibility or inflexibility transom extension (2066) and tubercle (2067), flexible connecting member (2060), outlet (2105), second housing component (2020), multiple power actuator exit (2200) and detection of analytes membranous system.The region comprising the part (2047) of detection of analytes membranous system has been exaggerated and has partly described a part for afterburning component (2070), test membrane (2045), absorption component (2055) and removable locking component extension (2068).

Figure 33 partly describes shell (2020), capillary channel (2301) and channel system (2300).A part of Figure 33 is exaggerated to describe pad (2050), absorption component (2055) and multiple kapillary (2301).

Figure 34 partly describes the sectional view that may be used for the device detecting multiple analytes with single signal, comprises multiple port (2036), entrance (2035), removable locking component (2065), the movable member (2100) of this removable locking component removable, afterburning component (2700), power actuator exit (2200), multiple absorption component (2055), test membrane (2045) and removable locking component extension (2068).Figure 34 also describes the exploded view of a part for detection of analytes membranous system, and this part comprises pad (2050), permeable membrane (2056) and absorption component (2055).

Figure 35 partly describes the limiting examples of removable locking component (2065) and removable locking component extension (2068).

Figure 36 partly describes external view and the internal view of the shell comprising multiple port (2036) and entrance (2035).

Figure 37 partly describes internal view and the external view of the shell comprising multiple power actuator exit (2200) and movable member outlet (2105).

Figure 38 partly describes the device comprising cylinder (3100), power actuator (3200) and power releasing device (3000) and outlet (3400) and the detection of analytes membranous system susceptor (3300) that can surround detection of analytes membranous system.

Figure 39 be partly depicted in describe in Figure 31 outlet (3400), susceptor (3300) and cylinder (3100) enlarged view.

Figure 40 partly describes the exploded view of the cylinder (3100) comprising the first casing component (3110), entrance (3135), pad (3350), second housing component (3120) and multiple film clamper (3122).

Figure 41 partly describes the device for detect analytes, and device comprises entrance (3335), membranous system susceptor (3300) and indicating meter (3500).

Figure 42 is partly depicted in the inside that may be used for the device detecting multiple analytes with single signal described in Figure 41.Device comprises the cylinder (3100), membranous system susceptor (3300), power actuator (3200), spectrometer (such as, optical pickup or the photodetector (3600), optionally pad remover (3201), optionally waste receptacle (3606), motor and membranous system susceptor thruster (3605/3607) that comprise detection of analytes membranous system.

Figure 43 is presented at the inside that may be used for the device detecting multiple analytes with single signal described in Figure 41 and Figure 42, and device is in the various stages used together with the same components described in Figure 35.Figure 43 A describes cylinder and is just inserting in susceptor.Figure 43 B describes the susceptor clamping just mobile cylinder for sample administration below entrance, and Figure 43 C description sample is just analyzed by spectrometer.

Figure 44 describes the exploded view that may be used for the device detecting multiple analytes with single signal, device comprises the first casing component (10), second housing component (20), groove (25) for sliding button, sliding button (30), import (35), test membrane (45), pad (50), other film (51), tackiness agent (52), multiple absorption component (such as, padding) (55), transom (60), locking component (65) and afterburning component (70).Can as described herein and/or shown assembled components can use the device of vertical current detect analytes to manufacture.

Figure 45 describes the decomposed figure that may be used for the device detecting multiple analytes with single signal, comprise the first casing component (10), second housing component (20), the groove (25) for sliding button, sliding button (30), import (35), the test membrane had no, pad (50), multiple absorption component (such as, padding) (not shown), transom (60), locking component (not shown) and afterburning component (not shown).Also can according to method manufacture described herein and other versions using this device.

Refer now to following examples and describe embodiment.These embodiments have been merely illustration purpose and provide and embodiment and never should be considered as being limited to these embodiments, but should be considered as containing and become significantly any and all changes form due to instruction provided herein.Those skilled in the art easily should identify multiple nonessential parameter, and parameter can through changing or revising to produce substantially similar result.

Embodiment

Embodiment 1:

Carry out two independently PCR reactions with shiga toxin gene as template, reaction produces by the amplicon of following mark: 1) digoxin and vitamin H, and 2) FITC and vitamin H.Amplicon mixes subsequently under Streptavidin (bridge-jointing unit) presence or absence, or enter in quick circulation analysis respectively: sample A) only amplicon 1, sample B) only amplicon 2, or sample C) there is and not have amplicon 1+ amplicon 2 under Streptavidin.Circulation analysis is made up of the solid support thing (nitrocellulose membrane) being coated with anti-digoxin (the first capture agent) and the colloidal gold particle that is coated with anti-FITC antibody.In this context, the sample C only with Streptavidin produces single positive test signal, and sample A and sample B or the sample C without Streptavidin produces negative test.

Embodiment 2: use amplicon bridge joint to detect multiple analytes.

Material:

PCR reagent: OneTaq Hot Start polysaccharase (New England's biology laboratory (New England Biolabs)); 5X standard reaction damping fluid; Haptenization MHALT1.RV (integrated DNA technique company (Integrated DNA Technologies) (IDT)); Haptenization MgC.CH1AS (IDT); INV018.7E4 V _hgene template (ZG); DNTP; DH ₂o.

Even variable Rate with 3-4 DEG C/s in standard temperature circulator carries out PCR.Run PCR reaction by vertical current analysis, analyze such as analysis described herein, comprise Veriflow Cassette (stealthy sentry company (Invisible Sentinel)).

Amplicon is produced according to standard scheme.A kind of amplicon of generation fluorescein isothiocyanate (FITC) and tetramethylrhodamin (TAMRA) double-tagging, and the another kind of amplicon of generation TAMRA and digoxin (DIG) double-tagging.DNA cloning from PCR reaction can be precipitated alternatively.If precipitated, so can be precipitated by EtOH or Virahol+1/10v sodium acetate 3M (pH5.2).In order to promote precipitation, 1uL tRNA glycogen also can be added.Carry out lasting minimum 2 hours at making to be deposited in-20 DEG C, or carry out lasting 15 minutes at-80 DEG C.The DNA of precipitation under top speed centrifugal about 15 minutes.Abandoning supernatant, makes DNA precipitate dry air 15 minutes.Optional secondary flushing can be carried out with the 70%EtOH that 20uL is ice-cold, centrifugal subsequently and dry.Suspension DNA is made to precipitate and make DNA at room temperature rehydrated about 24 hours with TE (Tris-HCl/EDTA).The amplicon produced is universal sequence and does not all have a specificity to any specific bacteria.

Amplicon with identify FITC biotinylated antibody and identify that the antibody of rhodamine (that is, TAMRA marks) mixes.Can mixtures incubated long period section, such as 5,10,15,20,25 or 30 minutes, but long period nonessential.The mixture hatched can add in Veriflow Cassette (vertical current device), and it comprises the test membrane containing unlabelled anti digoxin antibody and the pad containing Streptavidin-Jin binding substances.The existence of device bridge detection mixture, bridge joint mixture contains two kinds of amplicons with single signal (Radioactive colloidal gold).Suitably contrast and only Radioactive colloidal gold detected when producing the necessary all components of bridge joint mixture and all existing.Do not wish to be bound to any particular theory, Fig. 3 illustrates the mixture that can be formed with different components.When forming bridge joint mixture (see Fig. 3), Radioactive colloidal gold signal detected.Also the detectable signal of other types can be used.If the one in amplicon does not exist, so can't detect signal.After in sample access to plant, discharge Streptavidin-colloidal gold composite from pad and remove pad.How to produce and use the embodiment of vertical current device can be found in herein with US Patent No. 8,012,770, US 8,183,059 and U.S. patent application case US 13/500,997, US 13/360,528, US 13/445, in 233, each case is incorporated herein by reference in full.These results confirm, can detect two kinds of analytes specifically with single detectable signal (being Radioactive colloidal gold in this embodiment).The detection of signal do not depend on carry out PCR reactions steps after the precipitation of amplicon.

The disclosure of the every section of patent quoted herein, patent application case, publication and accession number are incorporated herein all by reference in full.

Although the present invention is disclosed with reference to particular implementation, it is evident that, other embodiments of the present invention and version can by those skilled in the art when not designing departing from when true spirit of the present invention and category.Following claims is intended to be interpreted as comprising all embodiments and equivalent variations.

Background technology

Detect multiple analytes usually to need to use multiple signal or multiple reaction, point or hole to determine whether sample has multiple analytes.This can make explanation become complicated, and when hotchpotch be classified as have two or more can detect feature, identification can be made for challenging final user.Therefore, in order to simplify and provide comprehensive qualitative report to final user, need the method and composition that can detect the multiple analytes in sample with single signal.The present invention meets the need and other needs.

Summary of the invention

The invention provides concurrently (side by side, concurrently) detect the method for the first analyte and the second analyte, comprising: solid support thing and the first analyte, the second analyte, the bridge-jointing unit that comprises the second capture agent are contacted with the detecting signal unit comprising the 3rd capture agent; With the presence or absence of detection signal detecting unit, described detecting signal unit indicates the presence or absence of the first analyte and the second analyte concurrently, and wherein the first capture agent fixes (adhere to, attached, affix) in solid support thing; First analyte comprises the first-phase mutual effect unit being incorporated into the first capture agent and the second-phase mutual effect unit being incorporated into bridge-jointing unit; And the second analyte comprise in conjunction with bridge-jointing unit first-phase mutual effect unit and be incorporated into the second-phase mutual effect unit of detecting signal unit.

The present invention also provides the method detecting the first analyte, the second analyte and the 3rd analyte with single signal concurrently, comprising: first, second, and third analyte is contacted with detecting signal unit with solid support thing, the first bridge-jointing unit, the second bridge-jointing unit; With the existence of detection signal detecting unit, detecting signal unit single signal indicates the existence of first, second, and third analyte concurrently, wherein: the first analyte comprises first-phase mutual effect unit and second-phase mutual effect unit; Second analyte comprises first-phase mutual effect unit and second-phase mutual effect unit; 3rd analyte comprises first-phase mutual effect unit and second-phase mutual effect unit; Solid support thing comprises the first capture agent of the first-phase mutual effect unit being incorporated into the first analyte; First bridge-jointing unit is incorporated into the second-phase mutual effect unit of the first analyte and the first-phase mutual effect unit of the second analyte; Second bridge-jointing unit is incorporated into the second-phase mutual effect unit of the second analyte and the first-phase mutual effect unit of the 3rd analyte; And detecting signal unit is incorporated into the second-phase mutual effect unit of the 3rd analyte.Interaction unit can be different from each other on each analyte.

In some embodiments, there is provided the method detecting the first analyte and the second analyte concurrently, method comprises: solid support thing and the first paid close attention to analyte, the second analyte paid close attention to, the bridge-jointing unit that comprises the second capture agent are contacted with the detecting signal unit comprising the 3rd capture agent; With the presence or absence of detection signal detecting unit, the presence or absence of the second analyte that described detecting signal unit indicates the first paid close attention to analyte concurrently and pays close attention to, wherein: the first capture agent is fixed on solid support thing; The first analyte of paying close attention to comprises the first-phase mutual effect unit being incorporated into the first capture agent and the second-phase mutual effect unit being incorporated into bridge-jointing unit; And the second analyte paid close attention to comprises the first-phase mutual effect unit in conjunction with bridge-jointing unit; Be incorporated into the second analyte, the first-phase mutual effect unit of the second analyte or second-phase mutual effect unit, the mixture of the first and second analytes or the detecting signal unit of the component of bridge-jointing unit that only exists when mixture contains the first and second analytes.

Also providing package is containing the mixture of solid support thing, the first analyte, the second analyte, bridge-jointing unit and detecting signal unit for embodiment described herein, and wherein each member of mixture is bonded to each other directly or indirectly.

Also providing package is containing the mixture of solid support thing, the first analyte, the second analyte, the 3rd analyte, the first bridge-jointing unit, the second bridge-jointing unit and detecting signal unit for embodiment described herein, and wherein solid support thing, the first analyte, the second analyte, the 3rd analyte, the first bridge-jointing unit, the second bridge-jointing unit and detecting signal unit are bonded to each other directly or indirectly.

The first analyte providing detection concurrently to pay close attention to herein and the method for the second analyte paid close attention to.In some embodiments, method comprises solid support thing and the first paid close attention to analyte, the second analyte paid close attention to, the bridge-jointing unit that comprises the second capture agent is contacted with the detecting signal unit comprising the 3rd capture agent; With the presence or absence of detection signal detecting unit, the presence or absence of the second analyte that described detecting signal unit indicates the first paid close attention to analyte concurrently and pays close attention to, wherein the first capture agent is fixed on solid support thing; The first analyte of paying close attention to comprises the first-phase mutual effect unit being incorporated into the first capture agent and the second-phase mutual effect unit being incorporated into bridge-jointing unit; And the second analyte paid close attention to comprises first-phase mutual effect unit and second-phase mutual effect unit, and wherein first-phase mutual effect unit is in conjunction with bridge-jointing unit; Be incorporated into following detecting signal unit: i) the second analyte, ii) the first-phase mutual effect unit of the second analyte or second-phase mutual effect unit, iii) component of mixture of the first and second analytes or iv) component of analyte-bridge joint mixture that only exists when mixture contains the first and second analytes.

In some embodiments, the first and second interaction unit of the first analyte paid close attention to and the first and second interaction unit of the second analyte paid close attention to are allos interaction unit independently of one another.In some embodiments, the second-phase mutual effect unit of the first analyte paid close attention to comprises identical allos interaction unit with the first-phase mutual effect unit of the second analyte paid close attention to.In some embodiments, the second-phase mutual effect unit of the first analyte paid close attention to comprises different allos interaction unit with the first-phase mutual effect unit of the second analyte paid close attention to.In some embodiments, the first-phase mutual effect unit of the first analyte paid close attention to comprises identical allos interaction unit with the second-phase mutual effect unit of the second analyte paid close attention to.In some embodiments, the first-phase mutual effect unit of the first analyte paid close attention to comprises different allos interaction unit with the second-phase mutual effect unit of the second analyte paid close attention to.

The method of the 3rd analyte detecting the first paid close attention to analyte, the second analyte paid close attention to concurrently with single signal and pay close attention to is provided.In some embodiments, method comprises first, second, and third paid close attention to analyte is contacted with detecting signal unit with solid support thing, the first bridge-jointing unit, the second bridge-jointing unit; With the existence of detection signal detecting unit, described detecting signal unit single signal indicates the existence of first, second, and third paid close attention to analyte concurrently, wherein: the first analyte paid close attention to comprises first-phase mutual effect unit and second-phase mutual effect unit; The second analyte of paying close attention to comprises first-phase mutual effect unit and second-phase mutual effect unit; The 3rd analyte paid close attention to comprises first-phase mutual effect unit and the 5th interaction unit; Solid support thing comprises the first capture agent of the first-phase mutual effect unit being incorporated into the first paid close attention to analyte; The second-phase mutual effect unit that first bridge-jointing unit is incorporated into the first paid close attention to analyte and the first-phase mutual effect unit of the second analyte paid close attention to; The second-phase mutual effect unit that second bridge-jointing unit is incorporated into the second paid close attention to analyte and the first-phase mutual effect unit of the 3rd analyte paid close attention to; And detecting signal unit is incorporated into: i) the 3rd analyte, ii) the first-phase mutual effect unit of the 3rd analyte or second-phase mutual effect unit, iii) component of first, second or the 3rd analyte complex or iv) component of analyte-bridge joint mixture that only exists when mixture contains first, second, and third analyte.

In some embodiments, bridge-jointing unit described herein is multivalence capture agent.In some embodiments, multivalence capture agent is immunoglobulin (Ig).In some embodiments, immunoglobulin (Ig) is IgM.Bridge-jointing unit can also be vitamin H.

Using appts single signal is provided to detect the method for multiple analytes concurrently.In some embodiments, method comprises the device and one or more sample contacts comprising multiple analytes that a) use single signal detection multiple analytes, wherein device comprises: shell, it comprises: with the import (inlet opening) of pad (binding substances pad, conjugate pad) in fluid contact; Afterburning component (force member); Contact the slidably locking component (slidable locking member) of afterburning component; Contact the transom (attachment member) of afterburning component; The sliding button (sliding button) of contact transom; With the detection membranous system (detection membrane system) comprising pad, test membrane (test membrane) and absorption component (absorbent member), pad, test membrane and absorption component substantially parallel to each other at least partially, afterburning member contact detects membranous system and can apply to be substantially perpendicular to the pressure detecting membranous system, sliding button moves slidably locking component, and pad comprises the detecting signal unit comprising the 3rd capture agent; Test membrane comprises the first capture agent being fixed on test membrane; Wherein one or more samples comprise the first paid close attention to analyte, the second analyte paid close attention to and the bridge-jointing unit comprising the second capture agent, the first wherein paid close attention to analyte comprises the first-phase mutual effect unit being incorporated into the first capture agent and the second-phase mutual effect unit being incorporated into bridge-jointing unit, and the second analyte paid close attention to comprises first-phase mutual effect unit in conjunction with bridge-jointing unit and second-phase mutual effect unit; The detecting signal unit wherein comprising the 3rd capture agent is incorporated into: i) the second analyte, ii) the first-phase mutual effect unit of the second analyte or second-phase mutual effect unit, iii) component of the first analyte and the second analyte complex or iv) component of analyte-bridge joint mixture that only exists when mixture contains the first and second analytes; And the presence or absence of b) detection signal detecting unit, the presence or absence of the second analyte that described detecting signal unit indicates the first paid close attention to analyte concurrently and pays close attention to.

In some embodiments, the part that method is included in one or more sample has contacted and pad has been moved after flowing through pad, make being exposed at least partially for detection signal detecting unit of test membrane thus, thus indicate the presence or absence of multiple analytes with single signal.In some embodiments, pad moves by making slidably locking component.In some embodiments, the first analyte and the second analyte are amplicon.In some embodiments, the first analyte and the second analyte are PCR reaction product.In some embodiments, the first-phase mutual effect unit of the first analyte is digoxin (digoxigenin) mark (label, label).In some embodiments, the second-phase mutual effect unit of the first analyte is rhodamine (rhodamine) mark.In some embodiments, the first-phase mutual effect unit of the second analyte is rhodamine mark.In some embodiments, the second-phase mutual effect unit of the second analyte is fluorescein-labelled.In some embodiments, the 3rd capture reagent bind is in the second-phase mutual effect unit of the second analyte.In some embodiments, the 3rd capture agent is biotinylated capture agent.In some embodiments, signal interaction unit is coated with Streptavidin (streptavidin).In some embodiments, signal interaction unit is the Radioactive colloidal gold (colloidal gold) of Streptavidin coating.In some embodiments, the first and second analytes are nucleic acid amplification product, wherein: the first analyte comprises digoxigenin labeled and rhodamine mark; Second analyte comprise rhodamine mark and fluorescein-labelled; First capture agent is the antibody of anti-digoxigenin labeled; Second capture agent is the antibody of anti-rhodamine mark; 3rd capture agent is biotinylated anti-fluorescein-labeled antibody; And signal interaction unit is the Radioactive colloidal gold of Streptavidin coating.

Accompanying drawing explanation

Fig. 1 especially explanation single signal detects the representativeness that two kinds of analytes carry out.

Fig. 2 especially explanation single signal detects the representativeness that three kinds of analytes carry out.

Fig. 3 especially illustrates just with two kinds of amplified productions that Radioactive colloidal gold detects.

Fig. 4 especially illustrates polycomponent bridge-jointing unit.

Fig. 5 especially illustrates and uses polycomponent bridge-jointing unit single signal to detect the representativeness that two kinds of analytes carry out.

Fig. 6 especially description taken in conjunction in the detecting signal unit of component of the bridge-jointing unit only existed when multiple analytes is present in mixture.

Fig. 7 especially explanation single signal detects the non-limiting workflow of multiple analytes.

Fig. 8 describes the skeleton view of representative device according to certain embodiments of the present invention.

Fig. 9 describes some assemblies of representative device according to certain embodiments of the present invention.

Figure 10 describes some assemblies of representative device according to certain embodiments of the present invention.

Figure 11 describes some assemblies of representative device according to certain embodiments of the present invention.

Figure 12 describes representative device some assemblies in various position according to certain embodiments of the present invention.

Figure 13: the side-view describing some assemblies of representative device according to certain embodiments of the present invention.

Figure 14 describes the side-view of some assemblies of representative device according to certain embodiments of the present invention.

Figure 15 A describes the side-view of some assemblies of representative device according to certain embodiments of the present invention.

Figure 15 B describe representative device according to certain embodiments of the present invention some assemblies (as but be not limited to inflexibility transom) view.

Figure 15 C describes the skeleton view of representative device according to certain embodiments of the present invention.

Figure 15 D describes the skeleton view of representative device according to certain embodiments of the present invention.

Figure 16 describes the flexible connecting member being connected to pad.

Figure 17 describes the film in representative casing component.

Figure 18 describes side-view and the top view of representative device according to certain embodiments of the present invention.

The alanysis quality testing that Figure 19 describes to be used for representative device according to certain embodiments of the present invention surveys membranous system.

The alanysis quality testing that Figure 20 describes to be used for representative device according to certain embodiments of the present invention surveys membranous system.

The alanysis quality testing that Figure 21 describes to be used for representative device according to certain embodiments of the present invention surveys membranous system.

The alanysis quality testing that Figure 22 describes to be used for representative device according to certain embodiments of the present invention surveys membranous system.

Figure 23 describes the afterburning component of representativeness of the representative device be used for according to certain embodiments of the present invention.

Figure 24 A-Figure 24 D describes representative device according to certain embodiments of the present invention.

Figure 25 A-Figure 25 C describes representative device according to certain embodiments of the present invention.

Figure 26 describes representative device according to certain embodiments of the present invention.

Figure 27 A-Figure 27 B describes the view of representative device according to certain embodiments of the present invention.

Figure 28 describes the fish-eye view of representative device according to certain embodiments of the present invention.

Figure 29 describes the exploded view of representative device according to certain embodiments of the present invention.

Figure 30 describes the internal view of representative device according to certain embodiments of the present invention.

Figure 31 A-Figure 31 B describes the sectional view of representative device according to certain embodiments of the present invention.

Figure 32 describes the exploded view of representative device according to certain embodiments of the present invention.

Figure 33 describes the internal view of representative device according to certain embodiments of the present invention.

Figure 34 describes the sectional view of representative device according to certain embodiments of the present invention.

Figure 35 describes the removable locking component of representativeness according to certain embodiments of the present invention.

Figure 36 describes representative shell according to certain embodiments of the present invention.

Figure 37 describes representative shell according to certain embodiments of the present invention.

Figure 38 A describes representative device according to certain embodiments of the present invention.

Figure 38 B describes representative device according to certain embodiments of the present invention.

Figure 39 describes the enlarged view of representative device according to certain embodiments of the present invention.

Figure 40 description cylinder according to certain embodiments of the present invention and the exploded view of detection of analytes membranous system.

Figure 41 describes representative device according to certain embodiments of the present invention.

Figure 42 describes representative device according to certain embodiments of the present invention.

Figure 43 A-Figure 43 C describes representative device according to certain embodiments of the present invention.

Figure 44 describes the exploded view of representative device according to certain embodiments of the present invention.

Figure 45 describes the exploded view of representative device according to certain embodiments of the present invention.

Claims (46)

1. detect a method for the first analyte paid close attention to and the second analyte paid close attention to concurrently, comprising:

Solid support thing and the first paid close attention to analyte, the second analyte paid close attention to, the bridge-jointing unit that comprises the second capture agent are contacted with the detecting signal unit comprising the 3rd capture agent; With

Detect the presence or absence of described detecting signal unit, the presence or absence of the second analyte that described detecting signal unit indicates the first described paid close attention to analyte concurrently and pays close attention to, wherein:

First capture agent is fixed on described solid support thing;

The first described paid close attention to analyte comprises the first-phase mutual effect unit being incorporated into described first capture agent and the second-phase mutual effect unit being incorporated into described bridge-jointing unit; And

The second described paid close attention to analyte comprises first-phase mutual effect unit and second-phase mutual effect unit, and wherein said first-phase mutual effect unit is in conjunction with described bridge-jointing unit;

Detecting signal unit, it is incorporated into: i) described second analyte, ii) the first-phase mutual effect unit of described second analyte or second-phase mutual effect unit, iii) component of mixture of described first analyte and described second analyte or iv) component of analyte-bridge joint mixture that only exists when described mixture contains described first analyte and described second analyte.

2. method according to claim 1, wherein, the described first-phase mutual effect unit of the described first-phase mutual effect unit of the first described paid close attention to analyte and second-phase mutual effect unit and the second described paid close attention to analyte and second-phase mutual effect unit are allos interaction unit independently of one another.

3. method according to claim 2, wherein, the described second-phase mutual effect unit of the first described paid close attention to analyte and the described first-phase mutual effect unit of the second described paid close attention to analyte comprise identical allos interaction unit.

4. method according to claim 2, wherein, the described second-phase mutual effect unit of the first described paid close attention to analyte and the described first-phase mutual effect unit of the second described paid close attention to analyte comprise different allos interaction unit.

5. method according to claim 2, wherein, the described first-phase mutual effect unit of the first described paid close attention to analyte and the described second-phase mutual effect unit of the second described paid close attention to analyte comprise identical allos interaction unit.

6. method according to claim 2, the described first-phase mutual effect unit of the first wherein said paid close attention to analyte and the described second-phase mutual effect unit of the second described paid close attention to analyte comprise different allos interaction unit.

7. method according to claim 1, the first wherein said paid close attention to analyte and the second described paid close attention to analyte are amplified production, peptide, sugar, antigen, nucleic acid molecule or their any combination independently.

8. method according to claim 1, wherein, the first described paid close attention to analyte and the second described paid close attention to analyte are amplified production.

9. method according to claim 1, wherein, described first-phase mutual effect unit be haptens and described first capture agent in conjunction with described haptenic molecule.

10. method according to claim 1, wherein, the described second-phase mutual effect unit of the first described paid close attention to analyte and the described first-phase mutual effect unit of the second described paid close attention to analyte are vitamin H and described second capture agent is compound in conjunction with vitamin H.

11. methods according to claim 10, wherein, described second capture agent is Streptavidin.

12. methods according to claim 1, wherein, described second-phase mutual effect unit be haptens and described 3rd capture agent in conjunction with described haptenic molecule.

13. methods according to claim 12, wherein, described 3rd capture agent is for being incorporated into described haptenic antibody.

14. methods according to claim 1, wherein, described detecting signal unit comprises radioactive labels, Radioactive colloidal gold, fluorescence labels, nanoparticle, radioactive nano particle, quantum dot, magnetic particle or enzyme.

15. 1 kinds of methods of the 3rd analyte detecting the first paid close attention to analyte, the second analyte paid close attention to concurrently with single signal and pay close attention to, comprising:

Described paid close attention to described first analyte, the second described paid close attention to analyte are contacted with detecting signal unit with solid support thing, the first bridge-jointing unit, the second bridge-jointing unit with the 3rd described paid close attention to analyte; With

Detect the existence of described detecting signal unit, described detecting signal unit single signal indicates the first described paid close attention to analyte concurrently, described pays close attention to the second analyte and described the existence paying close attention to the 3rd analyte, wherein:

The first described paid close attention to analyte comprises first-phase mutual effect unit and second-phase mutual effect unit;

The second described paid close attention to analyte comprises first-phase mutual effect unit and second-phase mutual effect unit;

The 3rd described paid close attention to analyte comprises first-phase mutual effect unit and the 5th interaction unit;

Described solid support thing comprises the first capture agent of the described first-phase mutual effect unit being incorporated into the first described paid close attention to analyte;

Described first bridge-jointing unit is incorporated into the described second-phase mutual effect unit of the first described paid close attention to analyte and the described first-phase mutual effect unit of the second described paid close attention to analyte;

Described second bridge-jointing unit is incorporated into the described second-phase mutual effect unit of the second described paid close attention to analyte and the described first-phase mutual effect unit of the 3rd described paid close attention to analyte; And

Described detecting signal unit is incorporated into the second-phase mutual effect unit of the 3rd described paid close attention to analyte.

16. methods according to claim 15, wherein, described first bridge-jointing unit is multivalence capture agent.

17. methods according to claim 16, wherein, described multivalence capture agent is immunoglobulin (Ig).

18. methods according to claim 17, wherein, described immunoglobulin (Ig) is IgM.

19. methods according to claim 15, wherein, described second bridge-jointing unit is incorporated into vitamin H.

20. methods according to claim 15, wherein, the described first-phase mutual effect unit of the first described paid close attention to analyte is haptens.

21. methods according to claim 15, wherein, the described second-phase mutual effect unit of the first described paid close attention to analyte is peptide.

22. methods according to claim 15, wherein, the described first-phase mutual effect unit of the second described paid close attention to analyte is peptide.

23. methods according to claim 15, wherein, the described second-phase mutual effect unit of the second described paid close attention to analyte is vitamin H.

24. methods according to claim 15, wherein, the described first-phase mutual effect unit of the 3rd described paid close attention to analyte is vitamin H.

25. methods according to claim 18, wherein, the described second-phase mutual effect unit of the 3rd described paid close attention to analyte is haptens.

26. 1 kinds of mixtures, comprise solid support thing, the first analyte paid close attention to, the second analyte, bridge-jointing unit and the detecting signal unit paid close attention to, wherein, each member of described mixture combines each other directly or indirectly.

27. mixtures according to claim 26, wherein:

Described solid support thing is incorporated into the first described paid close attention to analyte;

Described bridge-jointing unit is incorporated into the first described paid close attention to analyte and the second described paid close attention to analyte; And

Described detecting signal unit is incorporated into the second described paid close attention to analyte.

28. mixtures according to claim 26, wherein:

Described solid support thing comprises the first capture agent,

The first described paid close attention to analyte comprises first-phase mutual effect unit and second-phase mutual effect unit,

The second described paid close attention to analyte comprises first-phase mutual effect unit and second-phase mutual effect unit,

Described bridge-jointing unit comprises the capture agent of the described first-phase mutual effect unit of one or more described second-phase mutual effect unit being incorporated into the first described paid close attention to analyte independently and the second described paid close attention to analyte; And

Described detecting signal unit comprises the capture agent of the described second-phase mutual effect unit being incorporated into the second described paid close attention to analyte.

29. 1 kinds of mixtures, comprise solid support thing, the first analyte paid close attention to, the second analyte paid close attention to, the 3rd analyte, the first bridge-jointing unit, the second bridge-jointing unit and the detecting signal unit paid close attention to, wherein, described solid support thing, the first analyte paid close attention to, the second analyte paid close attention to, the 3rd analyte, the first bridge-jointing unit, the second bridge-jointing unit and the detecting signal unit paid close attention to combine each other directly or indirectly.

30. mixtures according to claim 29, wherein:

Described solid support thing is incorporated into described first analyte;

Described first bridge-jointing unit is incorporated into the first described paid close attention to analyte and the second described paid close attention to analyte;

Described second bridge-jointing unit is incorporated into the second described paid close attention to analyte and the 3rd described paid close attention to analyte; And

Described detecting signal unit is incorporated into the 3rd described paid close attention to analyte.

31. mixtures according to claim 29, wherein

Described solid support thing comprises the first capture agent;

The first described paid close attention to analyte comprises first-phase mutual effect unit and second-phase mutual effect unit;

The second described paid close attention to analyte comprises first-phase mutual effect unit and second-phase mutual effect unit;

The 3rd described paid close attention to analyte comprises first-phase mutual effect unit and second-phase mutual effect unit;

Described first bridge-jointing unit comprises the capture agent of the described first-phase mutual effect unit of one or more described second-phase mutual effect unit being incorporated into the first described paid close attention to analyte independently and the second described paid close attention to analyte;

Described second bridge-jointing unit comprises the capture agent of the described first-phase mutual effect unit of one or more described second-phase mutual effect unit being incorporated into the second described paid close attention to analyte independently and the 3rd described paid close attention to analyte; And

Described detecting signal unit comprises the capture agent of the described second-phase mutual effect unit being incorporated into the 3rd described paid close attention to analyte.

32. 1 kinds are detected the method for multiple analytes concurrently with single signal, and described method comprises:

I) device by single signal detection multiple analytes and one or more sample contacts comprising multiple analytes is used in,

Wherein said device comprises:

Shell, comprises:

With the import that pad is fluid contact;

Afterburning component;

Contact the slidably locking component of described afterburning component;

Contact the transom of described afterburning component;

Contact the sliding button of described transom;

With the detection membranous system comprising described pad, test membrane and absorption component,

Described pad, test membrane and absorption component substantially parallel to each other at least partially,

Detect membranous system described in described afterburning member contact and can apply to be substantially perpendicular to the pressure of described detection membranous system,

Slidably locking component described in described sliding button moves,

Described pad comprises the detecting signal unit comprising the 3rd capture agent;

Described test membrane comprises the first capture agent being fixed on described test membrane;

Wherein said one or more sample comprises the first paid close attention to analyte, the second analyte paid close attention to and the bridge-jointing unit comprising the second capture agent,

The first wherein said paid close attention to analyte comprises the first-phase mutual effect unit being incorporated into described first capture agent and the second-phase mutual effect unit being incorporated into described bridge-jointing unit; And the second described paid close attention to analyte comprises first-phase mutual effect unit in conjunction with described bridge-jointing unit and second-phase mutual effect unit;

The wherein said detecting signal unit comprising described 3rd capture agent is incorporated into described second analyte, the first-phase mutual effect unit of described second analyte or the component of second-phase mutual effect unit, described first analyte and described second analyte complex, or the component of the described bridge-jointing unit only existed when described mixture contains described first analyte and described second analyte; With

Ii) detect the presence or absence of described detecting signal unit, described detecting signal unit indicates the presence or absence of the first described paid close attention to analyte and the second described paid close attention to analyte concurrently.

33. methods according to claim 32, wherein, detect the part being included in described one or more sample contacted and described pad moved after flowing through described pad, make being exposed at least partially for detecting described detecting signal unit of described test membrane thus, thus indicate the presence or absence of described multiple analytes with single signal.

34. methods according to claim 32, wherein, described pad by described in making slidably locking component move.

35. methods according to claim arbitrary in claim 32 to 34, wherein, described one or more sample contacted with described pad before the described detection membranous system of compression.

36. methods according to claim arbitrary in claim 32 to 35, wherein, described first analyte and described second analyte are amplicon.

37. methods according to claim arbitrary in claim 32 to 36, wherein, described first analyte and described second analyte are PCR reaction product.

38. methods according to claim arbitrary in claim 32 to 37, wherein, the first-phase mutual effect unit of described first analyte is digoxigenin labeled.

39. methods according to claim arbitrary in claim 32 to 38, wherein, the second-phase mutual effect unit of described first analyte is rhodamine mark.

40. methods according to claim arbitrary in claim 32 to 39, wherein, the first-phase mutual effect unit of described second analyte is rhodamine mark.

41. methods according to claim arbitrary in claim 32 to 40, wherein, the second-phase mutual effect unit of described second analyte is fluorescein-labelled.

42. methods according to claim arbitrary in claim 32 to 41, wherein, described 3rd capture reagent bind is in the second-phase mutual effect unit of described second analyte.

43. methods according to claim arbitrary in claim 32 to 42, wherein said 3rd capture agent is biotinylated capture agent.

44. methods according to claim arbitrary in claim 32 to 43, wherein, described signal interaction unit is coated with Streptavidin.

45. methods according to claim arbitrary in claim 32 to 44, wherein, described signal interaction unit is the Radioactive colloidal gold of Streptavidin coating.

46. methods according to claim arbitrary in claim 32 to 35, wherein, described first analyte and described second analyte are nucleic acid amplification product, wherein:

Described first analyte comprises digoxigenin labeled and rhodamine mark;

Described second analyte comprise rhodamine mark and fluorescein-labelled;

Described first capture agent is anti-digoxigenin labeled antibody;

Described second capture agent is anti-rhodamine traget antibody;

Described 3rd capture agent is biotinylated anti-fluorescein labelled antibody; And

Described signal interaction unit is the Radioactive colloidal gold of Streptavidin coating.